Compositions and methods related to soluble G-protein coupled receptors (sGPCRs)

ABSTRACT

The present invention is directed to compositions and methods related to soluble G-protein coupled receptors (sGPCR). In ceratin aspects the invention includes compositions and methods related to a soluble corticotropin releasing factor receptor related protein, sCRFR2, as well as its effects on CRFR signaling and interaction between CRF family ligand and CRFR receptors, including but not limited to CRFR2, CRFR1 and functional or signaling capable variants thereof.

This application claims priority to provisional U.S. Patent ApplicationNo. 60/650,866, filed Feb. 8, 2005, which is incorporated herein byreference in its entirety.

The United States Government owns rights in present invention pursuantto grant number DK 26741 from the NIDDK.

I. TECHNICAL FIELD

The present invention is directed generally to method and compositionsrelated to molecular biology, neurology, and endocrinology. In certainaspects it is directed to compositions comprising and methods of usingsoluble G-protein coupled receptors (sGPCRs) as modulators of GPCRactivity and/or modulators of the pharmacologic effects of the ligandsthat bind such soluble GPCRs.

II. BACKGROUND OF THE INVENTION

Receptors, in general, are molecular structures located in the cellmembrane or within a cell that form a weak, reversible bond with anagent such as an antigen, hormone, or neurotransmitter. Each receptor isdesigned to bind with a specific agent(s). A specific family ofreceptors is the seven transmembrane (“7TM”) or G-Protein-CoupledReceptor (“GPCR”). These receptors link with a GuanineNucleotide-Binding G-protein (“G-protein”) in order to signal when anappropriate agent has bound the receptor. When the G-protein binds withGuanine DiPhosphate (“GDP”), the G-protein is inactive, or in an “offposition.” Likewise, when the G-protein binds with Guanine TriPhosphate(“GTP”), the G-protein is active, or in an “on position” wherebyactivation of a biological response in a cell is mediated.

GPCRs share a common structural motif. All these receptors have sevensequences of between 22 to 24 hydrophobic amino acids that form sevenalpha helices, each of which spans the membrane (i.e., transmembrane-1(TM-1), transmebrane-2 (TM-2), etc.). The transmembrane helices arejoined by strands of amino acids between transmembrane-2 andtransmembrane-3, transmembrane-4 and transmembrane-5, andtransmembrane-6 and transmembrane-7 on the exterior, or “extracellular”side, of the cell membrane (these are referred to as “extracellualrloops” or “extracellular” regions). The transmembrane helices are alsojoined by strands of amino acids between transmembrane-1 andtransmembrane-2, transmembrane-3 and transmembrane-4, andtransmembrane-5 and transmembrane-6 on the interior, or “intracellular”side, of the cell membrane (these are referred to as “intracellularloops” or “intracellular” regions). The “carboxy” (“C”) terminus of thereceptor lies in the intracellular space within the cell, and the“amino” (“N”) terminus of the receptor lies in the extracellular spaceoutside of the cell.

Generally, when a ligand binds with the receptor and “activates” thereceptor, there is a change in the conformation of the intracellularregion that allows for coupling between the intracellular region and anintracellular “G-protein.” It has been reported that GPCRs are“promiscuous” with respect to G-proteins, i.e., that a GPCR can interactwith more than one G-protein (Kenakin, 1988). Although other G-proteinsexist, currently, Gq, Gs, Gi, and Go are G-proteins that have beenidentified. Ligand-activated GPCR coupling with the G-protein begins asignaling cascade process or signal transduction. Under normalconditions, signal transduction ultimately results in cellularactivation or cellular inhibition. It is thought that the thirdintracellular loop (IC-3) as well as the carboxy terminus of thereceptor interact with the G-protein.

In general, the activity of almost every cell in the body is regulatedby extracellular signals. A number of physiological events in humans aswell as with a wide range of organisms use protein mediatedtransmembrane signaling via GPCRs. Signals from a specific GPCR causeactivation of a G-protein in the cell. The majority of signals aretransmitted by means of GPCRs into the cell interior. There are manyvarying aspects of this signaling process involving multiple receptorsubtypes for GPCRs and their G-protein linked counterparts as well as avariety of linked intracellular secondary messengers. The signaltransduction may result in an overall or partial activation orinactivation of an intracellular process or processes depending upon theproteins that are involved. Important signaling molecules orneurotransmitters which bind to GPCRs include, but are not limited tocorticotropin releasing factor, parathyroid hormone, morphine, dopamine,histamine, 5-hydroxytrytamine, adenosine, calcitonin, gastric inhibitorypeptide (GIP), glucagon, growth hormone-releasing hormone (GHRH),parathyroid hormone (PTH), PACAP, secretin, vasoactive intestinalpolypeptide (VIP), diuretic hormone, EMR1, latrophilin, brain-specificangiogenesis inhibitor (BAI), cadherin, EGF, LAG, (CELSR), and othersimilar proteins or molecules.

GPCRs constitute a superfamily of proteins. There are currently over2000 GPCRs reported in literature, which are divided into at least threefamilies: rhodopsin-like family (family A), the calcitonin receptors(family B), and metabotropic glutamate family (family C) (Ji et al.,1998). The reported GPCRs include both characterized receptors andorphan receptors for which ligands have not yet been identified. (Wilsonet al., 1999; Wilson et al., 1998; Marchese et al., 1999). Despite thelarge number of GPCRs, generally, each GPCR share a similar molecularstructure. Each GPCR comprises a string of amino acid residues ofvarious lengths. GPCRs lie within the cell membrane in seven distinctcoils called transmembranes. The amino terminus of the GPCR lies outsidethe cell with the extracellular loops, while the carboxy-terminus liesinside the cell with the intracellular loops.

The ligands for GPCRs comprise small molecules as well as peptides andsmall proteins. The interactions between these ligands and theirreceptors vary from system to system but they may require theinteraction with residues in several of the four extracellular domainsand the N-terminus. In some instances the N-terminus alone may maintainan ability to interact with or bind ligands. GPCRs with known ligandshave been associated with many diseases including multiple sclerosis,diabetes, rheumatoid arthritis, asthma, allergies, inflammatory boweldisease, several cancers, thyroid disorders, heart disease, retinitispigmentosa, obesity, neurological disorders, osteoporosis, HumanImmunodeficiency Virus (“HIV”) infection and Acquired Immune DeficiencySyndrome (“AIDS”) (Murphy et al., 2000; Mannstadt et al., 1999; Bergeret al., 1999; Jacobson et al., 1997; Meij, 1996;).

Accordingly, there is a need in the art for methods of producingmodulators of GPCRs and the ligands that bind GPCRs for use astherapeutics. These therapeutics may be used to prevent or treat GPCRassociated diseases and/or disorders.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods related toa sGPCR ligand binding domains, as well as effects of the sGPCR on GPCRsignaling and interaction between GPCR ligands and their GPCRs.

An embodiment of the invention includes an isolated soluble G-proteincoupled receptor (sGPCR) ligand binding domain. A sGPCR comprises all orpart of a GPCR extracellular domain. In one aspect of the invention thesGPCR is an soluble form of a GPCR family B member. In a further apsectthe sGPCR is a GPCR subfamily B1 member. In still further aspects, asGPCR is a soluble secretin receptor, VPAC₁ receptor, VPAC₂ receptor,PAC₁ receptor, glucagon receptor, growth hormone releasing hormone(GHRH) receptor, glucagon-related peptide 1 (GLP-1) receptor,glucagon-related peptide 2 (GLP-2) receptor, gastric inhibitorypolypeptide (GIP) receptor, corticotropin releasing factor 1 (CRF1)receptor, cortisotropin releasing factor 2 (CRF2) receptor, parathyroidhormone 1 (PTH1) receptor, parathyroid hormone 2 (PTH2) receptor,calcitonin receptor-like receptor, or calcitoinin receptor. The sGPCRcan be a soluble PTH1 receptor or PTH2 receptor. An embodiment of theinvention also includes a sGPCR that is a soluble form of thecorticotropin releasing factor receptor type 2α (sCRFR2α). The aminoacid sequence of a sCRFR2α may comprise an amino acid sequence encodedby exons 3, 4, and 5 of the CRFR2α gene or does not contain exon 6 orgreater. A recombinant sGPCR of the invention may include 75, 80, 85,90, 95, 100, 105, 110, 115, 120, 130, 135, 140, 150, 155, 160, 180, 200or more amino acids, including all ranges there between, of an GPCRextracellular domain(s), including all or part of the amino terminalextracellular domain. In certain apsects, a sGPCR ligand binding domainmay comprise an amino acid sequence at least 70, 75, 80, 85, 90, 95, or98% similar to 50, 75, 100, 125, 150 or more amino acids of SEQ ID NO:4(sCRFR2α), SEQ ID NO:8 (sCRFR2β), SEQ ID NO:12 (sCRFR2γ), or SEQ IDNO:15 (mCRFR2α). In a further aspect, a sCRFR comprises the amino acidsequence of SEQ ID NO:4, 8, 12, 15 or a combination thereof. In a stillfurther aspect, the invention includes an isolated sGPCR furthercomprising an affinity tag, a label, a detectable or therapeuticchemical moiety, a biotin/avidin label, a radionuclide, a detectable ortherapeutic enzyme, a fluorescent marker, a chemiluminescent marker, animmunoglobulin domain or any combination thereof. In one asepct, theGPCR comprises an immunoglobulin domain, in particular an Fc domain. ThesGPCR can be conjugated to a polymer, which includes, but is not limitedto polyethylene glycol (PEG).

Embodiments of the invention include polynucleotides encoding sGPCR ofthe invention. Polynucleotide may further comprise a promoter operablycoupled to the polynucleotide encoding the sGPCR. The sGPCR encodingsequence can be included in an expression cassette. The expressioncassette may be comprised in an expression vector. The expression vectormay include, but is not limited to a linear nucleic acid, a plasmidexpression vector, or a viral expression vector. In certain aspects, anexpression vector is comprised in a delivery vector, which may include,but is not limited to a liposome, a polypeptide, a polycation, a lipid,a bacterium, or a virus.

Still further embodiments of the invention include methods of modulatingthe activity of G-protien coupled receptor (GPCR) comprising: a)contacting a target tissue with a sGPCR; and b) binding a GPCR ligand inthe vicinity of the target tissue, wherein the activity of the GPCR inthe tissue is modulated. The ligand can be a GPCR family B ligand, aGPCR subfamily B1 ligand. In certain aspects the ligand is acorticotropin releasing factor (CRF), urocortin 1, urocortin 2,usorcortin 3, stresscopin, parathyroid hormone, PTH-related hormone,TIP39, calcitonin, amylin, CGRP (CALCA and CALCB), adrenomedullin,secretin, VIP, PACAP, glucagon, GHRH, GLP-1, GLP-2, GIP or anycombination thereof. The methods may also include contacting a targettissue comprising the steps of: a) preparing sGPCR ligand binding domainin an apprpriate pharmaceutical solution; and b) adminstering thepharmaceutical solution to an animal, human, subject, and/or patient inan amount to affect binding of a target ligand in the target tissue ofthe animal. Administration can be, but is not limited to ingestion,injection, endoscopy or perfusion. Injection includes, but is notlimited to intravenous, intramuscular, subcutaneous, intradermal,intracranial or intraperitoneal injection. Disorders that may betreated, ameliorated, modulation, reduced in severity, include disordersresulting from hyperactivation of a GPCR or hypersecretion of GPCRligand. In certain aspects the disorder is insulin sensitivity or typeII diabetes. The disorder may also include an anxiety-related disorder;a mood disorder; a post-traumatic stress disorder; supranuclear palsy;immune suppression; drug or alcohol withdrawal symptoms; inflammatorydisorders; pain; asthma; psoriasis and allergies; phobias; sleepdisorders induced by stress; fibromyalgia; dysthemia; bipolar disorders;cyclothymia; fatigue syndrome; stress-induced headache; cancer; humanimmunodeficiency virus infections; neurodegenerative diseases;gastrointestinal diseases; eating disorders; hemorrhagic stress;stress-induced psychotic episodes; euthyroid sick syndrome; syndrome ofinappropriate antidiarrhetic hormone; obesity; infertility; headtraumas; spinal cord trauma; ischemic neuronal damage; excitotoxicneuronal damage; epilepsy; cardiovascular and heart related disorders;immune dysfunctions; muscular spasms; urinary incontinence; seniledementia of the Alzheimer's type; multiinfarct dementia; amyotrophiclateral sclerosis; chemical dependencies and addictions; psychosocialdwarfism, insulin hypersensitivity or hyposensitivity, hypoglycemia,skin disorders; or hair loss. In certain aspects the disorder is ananxiety-related disorder; a mood disorder; bipolar disorder;post-traumatic stress disorder; inflammatory disorder; chemicaldependency and addiction; gastrointestinal disorder; or skin disorder.In a further aspect the anxiety-related disorder is generalized anxietyor the mood disorder is depression. In still a fruthre aspect thegastrointestinal disorder is irratable bowel syndrome.

Other embodiments of the invention include methods of detecting a GPCRligand comprising: a) contacting a sample suspected of containing a GPCRligand with a sGPCR polypeptide; and b) assessing the presence orabsence of sGPCR polypeptide bound ligand. The methods may furthercomprise characterizing the bound ligand. Characterizing a bound ligandincludes, but is not limited to various chromatographies, massspectrometry, peptide sequencing and the like. The sGPCR polypeptide mayor may not be operably coupled to a substrate or surface. The method canfurther comprise: c) adminstering a radiolabled GPCR ligand; and d)assessing binding or competition for binding of the radiolabeled GPCRligand to the sGPCR. The GPCR ligand may include, but is not limited tocorticotropin releasing factor (CRF), urocortin 1, urocortin 2,usorcortin 3, parathyroid hormone, PTH-related hormone, TIP39,calcitonin, amylin, CGRP (CALCA and CALCB), adrenomedullin, secretin,VIP, PACAP, glucagon, GHRH, GLP-1, GLP-2, or GIP.

Still other embodiments include methods of detecting a sGPCR comprising:a) contacting a sample suspected of containing a sGPCR with a ligandthat binds the sGPCR or a related surface bound GPCR; and b) assessingbinding of GPCR ligand with components of the sample. The method canfurther comprise characterizing the bound sGPCR, which can includechromatography, mass spectrometry, protein fragmentation and sequencing,and the like. A GPCR ligand may be operably coupled to a substrate orsurface. The methods can further compris: c) adminstering a radiolabledsGPCR; and d) assessing binding or competition for binding of theradiolabeled sGPCR to the GPCR ligand in the presence and absence of thesample being tested. Exemplary ligands include corticotropin releasingfactor (CRF), urocortin 1, urocortin 2, usorcortin 3, parathyroidhormone, PTH-related hormone, TIP39, calcitonin, amylin, CGRP (CALCA andCALCB), adrenomedullin, secretin, VIP, PACAP, glucagon, GHRH, GLP-1,GLP-2, GIP or other know GPCR ligands.

In yet still another embodiment of the invention includes antibodiesthat specifically bind a sGPCR. In certain aspects an antibody may bindthe amino terminus or carboxy terminus of the sGPCR. Aspects of theinvention include an antibody that binds a carboxy terminal 5, 10, 15,20 or more amino acid sequence, which may be derived from an alternativereading frame of a nucleotide sequenc that encodes a transmembraneregion of a GPCR (typically the result of alternative splicing and maybe engineered into a recombinant polynucleotide of the invention).

Embodiments of the invention include methods of detecting the expressionof a sGPCR, either using protein, nucleic acid or both protein andnucleic acid evaluation or assessment. Aspects of the invention includemethods of detecting a sGPCR mRNA comprising: a) obtaining a nucleicacid sample to be analyzed; and b) assessing the presence of a sGPCRnucleic acid comprising a splice junction resulting in a sGPCR. Themethod may include assessing the presence of a particular species ofmRNA by nucleic hybridization, nucleic acid amplification or othermethods of analyzing nucleic acids. In a particular aspect a sGPCR is asoluble B type GPCR, a soluble B1 type GPCR, a soluble CRFR, a sCRFR1, asCRFR2, or a sCRFR2α. A polynucleotide can include an exon/exon junctionthat includes the amino terminal amino acids of a GPCR and none or partof an exon encoding a portion of a transmembrane domain. In a particularaspect the splice junction of a sCRFR2α is an exon 5/exon 7 junction,wherein exon desigantion is based on the genomic designation of CRFR2exons. Based on the CRFR2α transcript the exons would be designated 3and 5, respectively (see FIG. 1 for an example).

A “soluble” GPCR (sGPCR) means a GPCR that comprises all or part of anextracellular domain of a receptor, but lacks all or part of one or moretransmembrane domains which normally retains the full length receptor inthe cell membrane, the soluble form is not integrated into the cellmembrane. Thus, for example, when such a soluble receptor is producedrecombinantly in a mammalian cell, it can be secreted frrom therecombinant host cell through the plasma membrane, rather than remainingat the surface of the cell. In general, a soluble receptor of theinvention is soluble in an aqueous solution. However, under certainconditions, the receptor can be in the form of an inclusion body, whichis readily solubilized by standard procedures. Such sGPCR may be derivedfrom an engneered nucleic acid, a processed protein (e.g., protealizedprotein), a synthesized protein, or an isolated splice variant. Apolynucleitide encoding such a sGPCR may be isolated or engineered.

As used herein, the terms “isolated” and “purified” are usedinterchangeably to refer to nucleic acids or polypeptides orbiologically active portions thereof that are substantially oressentially free from components that normally accompany or interactwith the nucleic acid or polypeptide as found in its naturally occurringenvironment. Thus, an isolated or purified nucleic acid or polypeptideis substantially free of other cellular material or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

An “isolated” nucleic acid is free of sequences (preferablyprotein-encoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acids can containless than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb ofnucleotide sequences that naturally flank the nucleic acids in genomicDNA of the cell from which the nucleic acid is derived.

As used herein, the term “isolated” or “purified” as it is used to referto a polypeptide of the invention means that the isolated protein issubstantially free of cellular material and includes preparations ofprotein having less than about 30%, 20%, 10%, 5% or less (by dry weight)of contaminating protein. When the protein of the invention orbiologically active portion thereof is recombinantly produced,preferably culture medium represents less than about 30%, 20%, 10%, or5% (by dry weight) of chemical precursors or non-protein-of-interestchemicals.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativeare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages, andobjects of the invention as well as others which will become clear areattained and can be understood in detail, more particular descriptionsand certain embodiments of the invention briefly summarized above areillustrated in the appended drawings. These drawings form a part of thespecification. It is to be noted, however, that the appended drawingsillustrate certain embodiments of the invention and therefore are not tobe considered limiting in their scope.

FIGS. 1A-1B. Illustrate an exemplary nucleotide and translated aminoacid sequence of a soluble GPCR, the CRF receptor type 2α (sCRFR2α)(FIG. 1A). Underlined amino acids indicate the unique C-terminal tail.Boxed residues indicate putative N-linked glycosylation sites. Schematicrepresentation of the structure of the mouse CRFR2 gene (upper panel),the two known functional transcripts in mouse, α and β (middle panels)and the novel sCRFR2α splice variant (lower panel) (FIG. 1B). Thelocations of the translation start sites (ATG) are indicated. Exonscoding for the N-terminal extracellular domain (ECD), the seventransmembrane domains (7TM), and the C-terminal cytoplasmic domain (CD)are indicated. 5′ and 3′-UTRs are indicated by hatched boxes. Blackboxes represent coding regions and open boxes represent exons downstreamto the stop codon.

FIGS. 2A-2C. Show expression of CRFR2α and sCRFR2α mRNA in mouse brainand pituitary. FIG. 2A is a schematic representation and theoligonucleotide primer locations of the amplified portion of mouseCRFR2α (upper panel) and sCRFR2α (lower panel) transcripts. Thelocations of the oligonuleotide primers, at exons three and seven, whichresult in the amplification of two products of 418 and 309 correspondingto CRFR2α and sCRFR2α, respectively, are indicated. FIG. 2B is arepresentative image of electrophoretic analysis of the semiquantitativeRT-PCR for mCRFR2α and sCRFR2α mRNA and the ribosomal protein S16 mRNA(upper panels). Southern blot hybridization of amplified mCRFR2α andsCRFR2α cDNA and the ribosomal protein S16 cDNA fragments were alsoperformed (lower panels). The radioactive bands were quantified byPhosphorImager and normalized values (relative to the S16 expression)are presented as relative densitometry units (FIG. 2C).

FIGS. 3A-3C. A highly specific antiserum raised in rabbit using asynthetic peptide fragment encoding the unique C-terminal tail of mousesCRFR2α protein (aa 113-143) was used to develop a sCRFR2αradioimmunoassay, used for immunoblot analysis and forimmunocytochemistry. FIG. 3A is a western immunoblot of mouse sCRFR2αisolated from the medium of COS-M6 cells transiently transfected withsCRFR2αFLAG construct reacted with anti-sCRFR2α-(113-143) serum (leftpanel) or monoclonal M2 anti-FLAG (right panel). Lanes 1, 2, and 3correspond to 0.1, 1.0, and 10 μl of sCRFR2α-FLAG extract, respectively.FIG. 3B, Displacement of [¹²⁵I]Tyr¹¹³ sCRFR2α (aa 113-143) binding torabbit anti-sCRFR2α (aa113-143) by synthetic sCRFR2α (aa 113-143) and bypurified COS-M6 expressed sCRFR2α (aa 113-143)-FLAG. FIG. 3C,Immunofluorescence staining of COS-M6 cells transiently transfected withmouse sCRFR2α construct visualized with the anti-sCRFR2α (aa 113-143)serum followed by a Cy3-conjugated secondary antibody (FIG. 3C(b)). Theslides were counterstained with DAPI to visualize both transfected andnon transfected cells (FIG. 3C(a)). Cells incubated with normal rabbitserum (NRS), as negative control, followed by a Cy3-conjugated secondaryantibody did not show any staining (FIG. 3C(c)).

FIGS. 4A-4G. Illustrates the presence of sCRFR2α-like immunoreactivity(ir) in the mouse brain using immunohistochemistry and radioimmunoassay(RIA). FIGS. 4A-4F show immunoperoxidase staining for sCRFR2α in selectmouse brain regions. Major sites of cellular expression included theprincipal output neurons of the olfactory bulb (FIG. 4A); the medialseptal nucleus (FIG. 4B); and the basolateral (BLA), but not the central(CeA) nucleus of the amygdala (FIG. 4C); cerebral cortex, where stainedcells were localized mainly in layers 5 and 2/3 (FIG. 4D); and rednucleus (FIG. 4E). In each of these sites, the pattern of cellularlabeling was similar, though not necessarily identical, to that of CRFR1mRNA expression. Immunolabeled fibers and varicosities were restrictedto a handful of cell groups, including the paraventricular nucleus ofthe hypothalamus (PVH; FIG. 4F). FIG. 4G, sCRFR2α-like immunoreactivityin acid-extracted and partially purified tissue isolated from mousebrain was measured by radioimmunoassay. Tissue extracts were tested at5-7 dose levels and displaced [¹²⁵I]-labeled Tyr¹¹³ sCRFR2α (aa 113-143)binding to rabbit anti-sCRFR2α (aa 113-143) in a dose-dependent manner.

FIGS. 5A-5B. sCRFR2α protein interferes with the induction of cAMP andMAPK signaling mediated by Ucn 1 or CRF. FIG. 5A shows activation ofCRE-luciferase reporter by Ucn 1 or CRF, with or without sCRFR2αpreincubation, in 293T cells transiently transfected with mouse CRFR2α.Luciferase reporter containing a fragment of the CRE promoter of theEVX1 gene was cotransfected into 293T cells with CRFR2α expressionvectors. Luciferase activity was measured following treatment (4 h) with0.0001-100 nM Ucn 1 or CRF, in the presence or absence of 0.1 nMsCRFR2α. Assays were normalized to cotransfected β-galactosidaseactivity. The representative means of six replicates from one experimentis shown in the graph. FIG. 5B, Equilibrated CATH.a cells were treatedwith Ucn 1 (10 nM) with or without sCRFR2α (0.4 or 4 nM). After 5 min ofreceptor stimulation, cell lysates were harvested and subjected toSDS-PAGE immunoblot analysis using phospho-ERK1/2-p42,44 antibody andERK2-p44 antibody. The ERK activation was calculated by normalizing thelevels of phosphorylated ERK1/2-p42,44 to total ERK2-p44. Therepresentative of means of triplicates from one experiment is shown inthe graph. *, P<0.05 vs. vehicle treatment, #, P<0.05 vs. Ucn 1treatment, UD=undetected.

DETAILED DESCRIPTION OF THE INVENTION

Useful therapeutic approaches for the treatment of diseases associatedwith GPCRs and associated signalling pathways include the inhibition ormodulation of the activation or inhibition of the GPCR. One approach isthe development of small molecule inhibitors, which are costly todevelop and bring to market. A drawback of the treatment with smallmolecule inhibitors or antagonists of GPCRs is the risk of toxicity,particularly with repeated application. Also, many GPCRs have no smallmolecule receptor antagonists. The development of a GPCR anatagonistthat is less costly and/or less toxic than small molecule inhibitors isworthwhile. Embodiments of the invention are directed to compositionsand methods related to soluble GPCR (sGPCR) ligand binding domains, aswell as its effects on GPCR signaling and interaction between GPCRligands and their GPCRs. sGPCRs may be used to antagonize the activationor inhibition of GPCRs in vitro and/or in vivo.

I. G-Protein Couples Receptors (GPCRs)

GPCRs constitute a superfamily of proteins, which are divided into threefamilies: rhodopsin-like family (family A), the calcitonin receptors(family B), and metabotropic glutamate family (family C) (Ji et al.,1998), each of which may further be divided into subfamilies. Thereported GPCRs include both characterized receptors and orphanreceptors, those for which ligands have not yet been identified (Wilsonet al., 1999; Wilson et al., 1998; Marchese et al., 1999). Despite thelarge number of GPCRs, generally, each GPCR share a similar molecularstructure. Each GPCR comprises a string of amino acid residues ofvarious lengths. GPCRs lie within the cell membrane in seven distinctcoils called transmembranes. The amino terminus of the GPCR is outsidethe cell as are the extracellular loops, while the carboxy-terminus isinside the cell with the intracellular loops.

GPCR family A (Rhodopsin like) includes, but is not limited to amine,peptide, hormone protein, rhodopsin, olfactory, prostanoid,nucleotide-like, cannabinoid, platelet activating factor,gonadotropin-releasing hormone, thyrotropin-releasing hormone andsecretagogue, melatonin, viral, lysosphingolipid and LPA (EDG),leukotriene B4 receptor and other similar receptor proteins.

GPCR family B (Secretin like) includes, but is not limited to receptorsfor calcitonin, corticotropin releasing factor (CRF), gastric inhibitorypeptide (GIP), glucagon, growth hormone-releasing hormone (GHRH),parathyroid hormone (PTH), pituitary adenylate cyclase-activatingpolypeptide (PACAP), secretin, vasoactive intestinal polypeptide (VIP),diuretic hormone, EMR1, latrophilin, brain-specific angiogenesisinhibitor (BAI), methuselah-like proteins (MTH), cadherin/EGF/LAG(CELSR), and other similar ligands. Harmar (2001) describes threesubfamilies of GPCR family B, subfamily B1, B2 and B3.

Subfamily B1—Subfamily B1 includes, but is not limited to the classicalhormone receptors, which are encoded by at least 15 genes in humans,with at least five putative members in Drosophila and three in C.elegans. The ligands for receptors in this family are polypeptidehormones of approximately 27-141 amino-acid residues; at least nine ofthe mammalian receptors respond to ligands that are structurally relatedto one another (glucagon, glucagon-like peptides (GLP-1, GLP-2),glucose-dependent insulinotropic polypeptide, secretin, vasoactiveintestinal peptide (VIP), PACAP, and growth-hormone-releasing hormone(GHRH). All members of this subfamily have been shown to be capable ofregulating intracellular concentrations of cAMP by coupling to adenylatecyclase through a stimulatory G protein (Gs). Some members of thesubfamily are capable of signaling through additional G-protein-coupledsignaling pathways, for example through activation of phospholipase C.

Subfamily B2—Subfamily B2 consists of a large number of family-B GPCRswith long extracellular amino termini, containing diverse structuralelements linked to the core 7TM motif. The prototype members of thissubfamily were an EGF-module-containing, mucin-like hormone receptor(EMR1) isolated from a human neuroectodermal cDNA library (Baud et al.,1995) and the leukocyte cell-surface antigen CD97 (Hamann et al., 1995).Subfamily B2 also includes the calcium-independent receptors forα-latrotoxin. Three genes encoding calcium-independent latrotoxinreceptors (CL-1 CL-2 and CL-3) have been identified. Secondly, thebrain-specific angiogenesis inhibitors 1, 2 and 3 (BAI1, BAI2, BAI3), agroup of proteins that have been implicated in the vascularization ofglioblastomas are also included in this subfamily. Thirdly, the proteinencoded by the Drosophila gene flamingo and its orthologs in humans (thecadherin EGF LAG seven-pass G-type receptors Celsr1, Celsr2 and Celsr3)and in C. elegans (F15B9.7) is also included in the B2 subfamily.Finally, the subfamily includes a fourth, diverse group of receptorsthat contain some motifs common to receptors in subfamily B2 but areotherwise structurally unrelated (human epididymis 6 (HE6),EGF-TM7-latrophilin-related protein (ETL), theimmunoglobulin-repeat-containing receptor Ig hepta, G-protein-coupledreceptor 56 (GPR56) and very large G-protein-coupled receptor 1(VLGR1)). Analysis of the sequenced human genome (1 Apr. 2001, UCSCHuman Genome Project Working Draft (genome.ucsc.edu)) indicates thatthere are at least 18 human genes encoding members of subfamily B2, andthere are at least four in Drosophila and three in C. elegans. Thestructure and functions of members of subfamily B2 have been reviewedrecently by Stacey et al. (2000).

Subfamily B3—The prototype of a third group (subfamily B3) of family-BGPCRs is methuselah (mth), a gene isolated in a screen for single-genemutations that extended average lifespan in D. melanogaster (Lin et al.,1998). The gene encodes a polypeptide that displays sequence similarityto other family-B GPCRs solely within the TM7 region. A least eightparalogs of methuselah are encoded within the Drosophila genomesequence.

The characteristic feature of all family-B GPCRs is the 7TM motif, whichis distantly related to comparable regions of some other GPCR familiesbut much more highly conserved within family B. Conserved cysteineresidues within extracellular loops EC1 and EC2 probably form adisulphide bridge, by analogy with family-A GPCRs in which this featureis also conserved (Palczewski et al., 2000). In contrast to family-AGPCRs, however, many of which appear to rely on internal hydrophobicsequences for targeting to the plasma membrane, most family-B GPCRsappear to have an amino-terminal signal peptide. Studies usingsite-directed mutagenesis and the construction of chimeras betweenhormone receptors in family B have shown that the amino-terminalextracellular domain is essential for ligand binding but that thetransmembrane domains and associated extracellular loop regions of thereceptors provide information necessary for specific interaction withligands. All of the hormone receptors in family B contain a conservedregion within the amino-terminal extracellular domain close to TM1 thatmay play a role in ligand binding. Splice variation in this region ofthe PAC1 receptor has been shown to influence ligand-binding specificityand affinity (Dautzenberg et al., 1999).

Receptors in subfamily B2 contain a variety of additional structuralmotifs in their large amino-terminal extracellular domains that suggesta role for this domain in cell-cell adhesion and signaling. Theseinclude EGF domains (in Celsr1, Celsr2, Celsr3, EMR1, EMR2, EMR3, CD97and Flamingo), laminin and cadherin repeats (in Flamingo and its humanorthologs Celsr1, Celsr2 and Celsr3), olfactomedin-like domains (in thelatrotoxin receptors), thrombospondin type 1 repeats (in BAI1, BAI2 andBAI3) and, in Ig hepta, an immunoglobulin C-2-type domain also found infibroblast growth factor (FGF) receptor 2 and in the neural celladhesion molecule L1. VLGR1 has two copies of a motif (Calx-beta)present in Na+-Ca2+exchangers and integrin subunit β4.

Family C (Metabotropic glutamate/pheromone) GPCR includes Metabotropicglutamate, calcium-sensing like, putative pheromone receptors, GABA-B,orphan GPRC5, orphan GPCR6, bride of sevenless proteins (BOSS), tastereceptors (T1R) and other similar proteins.

In certain embodiments, the sGPCRs of the invention are class Breceptors. In aspect, the sGPCRs of the invention are subfamily B1receptors, and in a further aspect, the sGCPRs are CRFR1 and CRFR2, andparathyroid hormone receptor. Table 1 includes a non-limiting set ofexemplary members of the GPCR family, accession numbers and associatedUNIGENE and OMIM entries are incorporated herein by reference as of thepriority date and the date of filing of this application. Unigeneentries can be accessed by internet links contained in OMIM webpage.Numerous other GPCRs and their accession numbers may be found at thewebsite defined by the following address on the world wide webgpcr.org/7tm/htmls/entries.html. TABLE 1 Exemplary GPCRs. Protein acc.#/mRNA ace #/ OMIM # (each of which are GPCR description incorporated byreference) BRAIN-SPECIFIC ANGIOGENESIS INHIBITOR 1 (BAI 1)O14514/AB005297/602682 BRAIN-SPECIFIC ANGIOGENESIS INHIBITOR 2 (BAI 2)O60241/CR623649/602683 BRAIN-SPECIFIC ANGIOGENESIS INHIBITOR 3(KIAA0550a)(BAI 3) O60242/AB005299/602684 CALCITONIN RECEPTOR (CT-R,CALCR) P30988/NM_001742/114131 LEUCOCYTE ANTIGEN CD97P48960/X84700/601211 CALCITONIN GENE-RELATED PEPTIDE TYPE 1 RECEPTOR,CGRP Q16602/NM_005795/114190 TYPE 1 RECEPTOR, CALCRL, CGRPRCORTICOTROPIN RELEASING FACTOR RECEPTOR 1 (CRF-R, CRF1,P34998/NM_004382/122561 CRHR1, CRHR, CRFR) CORTICOTROPIN RELEASINGFACTOR RECEPTOR 2 (CRF-R, CRF2, Q13324/NM_001883/602034 CRHR2, CRF2R,CRH2R) CELL SURFACE GLYCOPROTEIN EMR1 (EMR1 HORMONE Q14246/X81479/600493RECEPTOR) EGF-LIKE MODULE EMR2 AAF21974/AF114491/606100 EGF-LIKEMODULE-CONTAINING MUCIN-LIKE RECEPTOR EMR3 AAK15076/AF239764/606101GASTRIC INHIBITORY POLYPEPTIDE RECEPTOR (GIP-R, GLUCOSE-P48546/NM_000164/137241 DEPENDENT INSULINOTROPIC POLYPEPTIDE RECEPTOR)GLUCAGON-LIKE PEPTIDE 1 RECEPTOR (GLP-1 RECEPTOR, GLP-1-R,P43220/NM_002062/138032 GLP1R) GLUCAGON RECEPTOR (GL-R, GCGR)P47871/NM_000160/138033 GLUCAGON-LIKE PEPTIDE 2 RECEPTOR (GLP-2RECEPTOR, GLP-2-R, O95838/NM_004246/603659 GLP-2R, GLP2R) GPROTEIN-COUPLED RECEPTOR 56 AAD30545/NM_005682/604110 GROWTHHORMONE-RELEASING HORMONE RECEPTOR (GHRH Q02643/NM_000823/139191RECEPTOR, GRF RECEPTOR, GRFR, GHRHR) PITUITARY ADENYLATE CYCLASEACTIVATING POLYPEPTIDE P41586/NM_001118/102981 TYPE I RECEPTOR (PACAPTYPE I RECEPTOR, PACAP-R-1, ADCYAP1R1) PARATHYROID HORMONE RECEPTOR(PTH2) P49190/NM_005048/601469 PARATHYROID HORMONE/PARATHYROIDHORMONE-RELATED Q03431/NM_000316/168468 PEPTIDE RECEPTOR (PTHR1)SECRETIN RECEPTOR (SCT-R, SCTR) P47872/NM_002980/182098 VASOACTIVEINTESTINAL POLYPEPTIDE RECEPTOR 1 (VIPR1, P32241/NM_004624/192321 VPAC1)VASOACTIVE INTESTINAL POLYPEPTIDE RECEPTOR 2 (VIPR2,P41587/NM_003382/601970 VIP2R, VPAC2, etc.)

A. Corticotropin Releasing Factor (CRF) and its Receptors

As an example of GPCRs contemplated by the invention, the CRF receptorsare described in detail. One of skill in the art would be able to adaptthese specific teachings to other members of the GPCR family,particularly type B and more particularly to subfamily B1 receptors. Incertain aspects the invention includes, but is not limited to the sGPCRderived from the soluble corticotropin releasing factor receptors(sCRFR), in particular sCRFR2α. The hypothalamic hypophysiotropicpeptide corticotropin releasing factor (CRF), originally isolated fromthe hypothalamus (Vale et al., 1981), plays an important role in theregulation of the hypothalamo-pituitary-adrenal (HPA) axis under basaland stress conditions (River and Vale, 1983; Muglia et al., 1995).Further, CRF acts to integrate endocrine, autonomic, and behavioralresponses to stressors (River and Vale, 1983; Muglia et al., 1995; Kooband Heinrichs, 1999). The mammalian CRF peptide family comprisesurocortin 1 (Ucn 1) (Vaughan et al., 1995) and the peptides, urocortin 2(Ucn 2) and urocortin 3 (Ucn 3) also known as stresscopin-relatedpeptide (Reyes et al., 2001; Hsu and Hsueh, 2001), and stresscopin (Hsuand Hsueh, 2001; Lewis et al., 2001), respectively.

The effects of CRF-related peptides are mediated through activation oftwo high affinity membrane receptors, CRFR1 (Chen et al., 1993; Vita etal., 1993; Chang et al., 1993) and CRFR2 (Perrin et al., 1995; Stenzelet al., 1995; Kishimoto et al., 1995; Lovenberg et al., 1995; Chen etal., 2005), which belong to the B1 subfamily of seven-transmembranedomain (7TMD) receptors that signal by coupling to G-proteins. Onefunctional variant of the CRFR1 gene is expressed both in humans androdents, along with several non-functional variants, which are producedby differential splicing of various exons (Pisarchik and Slominski,2004; Grammatopoulos et al., 1999). The CRFR2 has three functionalsplice variants in human (α, β, and γ) and two rodent variants (α and β)that are produced by the use of alternate 5′ exons (Perrin et al., 1995;Stenzel et al., 1995; Kishimoto et al., 1995; Lovenberg et al., 1995;Chen et al., 2005; Grammatopoulos et al., 1999; Kostich et al., 1998).CRFR1 mRNA is widely expressed in mammalian brain and pituitary, withhigh levels found in the anterior pituitary, cerebral cortex,cerebellum, amygdala, hippocampus, and olfactory bulb (Van Pett et al.,2000). In the periphery, CRFR1 is expressed in testes, ovary, skin, andspleen. CRFR2 mRNA is expressed in a discrete pattern in the brain withhighest densities in the lateral septal nucleus (LS), bed nucleus ofstria terminalis (BNST), ventromedial hypothalamic nucleus (VMH),olfactory bulb, and mesencephalic raphe nuclei (Van Pett et al., 2000).The CRFR2α is the major splice variant expressed in the rodent brain(Lovenberg et al. 1995) while CRFR2 is expressed in peripheral tissues,with highest levels in the skeletal muscle, heart, and skin (Perrin etal., 1995).

The distributions of CRFR1 and CRFR2 are distinct and imply diversephysiological functions, as demonstrated by the divergent phenotypes ofthe CRFR1 or CRFR2 null mice. Mice deficient for CRFR1 display decreasedanxiety-like behavior and have an impaired stress response (Smith etal., 1998; Timpl et al., 1998), while the CRFR2-null mice have increasedanxiety-like behaviors and an exaggerated HPA response to stress (Zhu etal., 1999; Valerio et al., 2001; Khan et al., 1993). However, theresponses to administration of CRFR2 agonists and antagonists intospecific brain regions reveal both anxiolytic and anxiogenic roles forCRFR2 (Bale and Vale, 2004).

Radioreceptor and functional assays have demonstrated that CRFR1 andCRFR2 differ pharmacologically: Ucn 1 has equal affinities for bothreceptors and is more potent than CRF on CRFR2, whereas Ucn 2 and Ucn 3appear to be selective for CRFR2 (Vaughan et al, 1995; Reyes et al.,2001; Lewis et al., 2001). The activation of specific CRFRs in distincttissues or cell types by receptor-selective CRF peptides initiates avariety of signaling pathways, including coupling to differentG-proteins, stimulation of PKB, PKC, intracellular calcium, andmitogen-activated protein kinase (MAPK) (for reviews see Bale and Vale,2004; Perrin and Vale, 1999; Brar et al., 2002).

CRFR1 and CRFR2 both exist as multiple splice variants. The inventorshave identified a cDNA from mouse brain encoding an exemplary splicevariant of sCRFR2α in which exon six is deleted from the nucleic acidencoding CRFR2α. Translation of this isoform produces a predicted 143amino acid soluble protein. The translated protein includes a majorityof the first extracellular domain (ECD1) of the CRFR2α followed by aunique 38 amino acid hydrophilic C-terminus resulting from a frame shiftproduced by deletion of exon six. Studies have demonstrated high levelsof expression of sCRFR2α in the olfactory bulb, cortex, and midbrainregions. A protein corresponding to sCRFR2α, expressed and purified fromeither mammalian or bacterial cell systems, binds several CRF familyligands with low nanomolar affinities. Further, the purified sCRFR2αprotein inhibits cellular responses to CRF and urocortin 1. Thus, asCRFR2α protein can be a biological modulator of CRF family ligands. Themodulation of CRF family ligands is not limited to brain and may be usedin any tissue that is exposed to one or more members of the CRF familyof ligands.

Aspects of the invention generally relate to compositions and methods ofachieving a therapeutic effect, including the modulation of GPCR ligandactivity, such as CRF family ligands, using a soluble GPCR ligandbinding polypeptide, such as CRF binding polypeptide, as an antagonisteither alone or together with one or more other hormone antagonist(e.g., small molecule antagonist), including but not limited toantagonist of ligand(s) of the CRF family.

One manner in which to antagonize the action of a ligand is to subjectthe ligand to a decoy or soluble receptor so as to limit the localconcentration of ligand(s) that bind the decoy and modulate the ligandsability to signal via its cell surface receptor. Soluble proteinsrelated to membrane receptors can be generated by enzymatic truncationof membrane bound receptors as suggested for the GHRH receptor (Rekaskiet al., 2000), dopamine D3 receptor (Liu et al., 1994), and calcitoninreceptor (Seck et al., 2003), or by alternative splicing in the case ofthe glutamate receptors (Malherbe et al., 1999; Zhu et al., 1999;Valerio et al., 2001). Splice variants containing only the extracellularregion of GPCRs have been reported (Pisarchik and Slominski, 2004;Grammatopoulos et al., 1999; Kostich et al., 1998; Malherbe et al.,1999; Zhu et al., 1999; Valerio et al., 2001; Khan et al., 1993; Graveset al., 1992; You et al., 2000; Schwarz et al., 2000). In the majorityof cases, these proteins act as binding, non-signaling molecules alsoreferred to as decoy receptors. Two partial cDNA fragments (CRFR1e andCRFR1h), comprising deletion of exon 3 and 4, and addition of a crypticexon in CRFR1 were identified in human skin and predicted to exist as asoluble proteins (Pisarchik and Slominski, 2004). One of thesefragments, CRFR1e, exhibited dominant negative effects whenco-transfected with the wildtype CRFR1.

Kehne and Lombaert (2002) disucuss non-peptidic CRF receptor antagonistsfor the treatment of anxiety, depression, and stress disorders. CRF isimplicated in psychiatric disorders, such as anxiety and depression.Since the identification of corticotropin releasing factor (CRF) anextensive research effort has solidified the importance of this 41 aminoacid peptide and its related family members in mediating the body'sbehavioral, endocrine, and autonomic responses to stress.

Preclinical and clinical evidence implicate CRF, in general, and CRFreceptors, in particular, in anxiety and depression. Clinical studieshave demonstrated a dysfunctional hypothalamic-pituitary-adrenal (HPA)axis and/or elevated CRF levels in depression and in some anxietydisorders. Preclinical data utlilizing correlational methods, geneticmodels, and exogenous CRF administration techniques in rodents andnon-human primates supports a link between hyperactive CRF pathways andanxiogenic and depressive-like symptoms. Studies employing the use ofreceptor knockouts and selective, non-peptidic antagonists of the CRFR1have demonstrated anxiolytic and antidepressant effects under certaintypes of laboratory conditions. A Phase II, open-label, clinical trialin major depressive disorder has reported that a CRFR1 antagonist wassafe and effective in reducing symptoms of anxiety and depression.

Various nonlimiting activities of CRF antagonists are described by Owenset al. (1991). CRF antagonists are described as being effective in thetreatment of stress-related illnesses; mood disorders such asdepression, major depressive disorder, single episode depression,recurrent depression, child abuse induced depression, postpartumdepression, dysthemia, bipolar disorders, and cyclothymia; chronicfatigue syndrome; eating disorders such as anorexia and bulimia nervosa;generalized anxiety disorder; panic disorder; phobias;obsessive-compulsive disorder; post-traumatic stress disorder; painperception such as fibromyalgia; headache; gastrointestinal diseases;hemorrhagic stress; ulcers; stress-induced psychotic episodes; fever;diarrhea; post-operative ileus; colonic hypersensitivity; irritablebowel syndrome; Crohn's disease; spastic colon; inflammatory disorderssuch as rheumatoid arthritis and osteoarthritis; pain; asthma;psoriasis; allergies; osteoporosis; premature birth; hypertension,congestive heart failure; sleep disorders; neurodegenerative diseasessuch as Alzheimer's disease, senile dementia of the Alzheimer's type,multiinfarct dementia, Parkinson's disease, and Huntington's disease;head trauma; ischemic neuronal damage; excitotoxic neuronal damage;epilepsy; stroke; spinal cord trauma; psychosocial dwarfism; euthyroidsick syndrome; syndrome of inappropriate antidiuretic hormone; obesity;chemical dependencies and addictions; drug and alcohol withdrawalsymptoms; infertility; cancer; muscular spasms; urinary incontinence;hypoglycemia and immune dysfunctions including stress induced immunedysfunctions, immune suppression, and human immunodeficiency virusinfections; and stress-induced infections in humans and animals. Theseand other conditions amenable to CRF modulation are set out in theliterature, that includes Lovenberg et al. (1995); Chalmers et al.(1996); and U.S. Pat. No. 5,063,245, each of which is incorporated inits entirety by reference.

II. Polypeptides

Polypeptides of the invention include soluble forms of GPCRs or solublereceptors. Soluble receptors of the invention may comprise subunitswhich have been changed from a membrane bound to a soluble form. Thus,soluble peptides may be produced by truncating the polypeptide toremove, for example, the 7 transmembrane regions and/or the cytoplasmictail. Alternatively, the transmembrane domains may be abolished bydeletion, or by substitutions of the normally hydrophobic amino acidresidues which comprise a transmembrane domain with hydrophilic ones. Ineither case, a substantially hydrophilic or soluble polypeptide iscreated which will reduce lipid affinity and improve aqueous solubility.Deletion of the transmembrane domains is preferred over substitutionwith hydrophilic amino acid residues because it avoids introducingpotentially immunogenic epitopes. Soluble receptors of the invention mayinclude any number of well-known leader sequences at the N-terminus.Such a sequence would allow the peptides to be expressed and targeted tothe secretion pathway in a eukaryotic system.

A. Fusion Proteins

Receptors are powerful tools to elucidate biological pathways and totreat various disease states via their easy conversion to immunoglobulinfusion proteins. These dimeric soluble receptor forms are goodinhibitors of events mediated by either secreted or surface boundligands. By binding to these ligands they prevent the ligand frominteracting with cell associated receptors. Not only are thesereceptor-Ig fusion proteins useful in an experimental sense, but theyhave been successfully used clinically in the case of TNF-R-Ig to treatinflammatory bowel disease, rheumatoid arthritis, and the acute clinicalsyndrome accompanying OKT3 administration (Eason et al., 1996; vanDullemen et al., 1995). The inventors contemplate that manipulation ofthe many events mediated by signaling through the GPCRs will have wideapplication in the treatment of GPCR associated diseases.

Preferably, stable plasma proteins—which typically have a half-lifegreater than hours in the circulation of a mammal—can be used toconstruct the receptor fusion proteins. Such plasma proteins include butare not limited to: immunoglobulins, serum albumin, lipoproteins,apolipoproteins and transferrin. Sequences that can target the solublereceptors to a particular cell or tissue type may also be attached tothe receptor ligand binding domain to create a specifically localizedsoluble receptor fusion protein.

All or a functional fragment of GPCR extracellular region comprising theGPCR ligand binding domain may be fused to an immunoglobulin constantregion like the Fc domain of a human IgG1 heavy chain. Solublereceptor-IgG fusions proteins are common immunological reagents andmethods for their construction are well known in the art (see, forexample U.S. Pat. No. 5,225,538, which is incorporated herein in itsentirety by reference).

A functional GPCR ligand binding domain may be fused to animmunoglobulin (Ig) Fc domain. The Ig Fc may be derived from animmunoglobulin class or subclass including but not limited to IgG1. TheFc domains of antibodies belonging to different Ig classes or subclassescan activate diverse secondary effector functions. Activation occurswhen the Fc domain is bound by a cognate Fc receptor. Secondary effectorfunctions include the ability to activate the complement system or tocross the placenta. The properties of the different classes andsubclasses of immunoglobulins are described in the art.

One skilled in the art will appreciate that different amino acidresidues forming the junction point of the receptor-Ig fusion proteinmay alter the structure, stability and ultimate biological activity ofthe sGPCR fusion protein. One or more amino acids may be added to theC-terminus of the selected sGPCR fragment to modify the junction pointwith the selected fusion domain.

The N-terminus of the sGPCR fusion protein may also be varied bychanging the position at which the selected sGPCR DNA fragment iscleaved at its 5′ end for insertion into the recombinant expressionvector. The stability and activity of each sGPCR fusion protein may betested and optimized using routine experimentation, including but notlimited to assays for ligand binding.

Using sGPCR ligand binding domain sequences within the extracellulardomain as shown herein, amino acid sequence variants may also beconstructed to modify the affinity of the sGPCR molecules for theirligands. The soluble molecules of this invention can compete for bindingwith endogenous receptors. It is envisioned that any soluble moleculecomprising a GPCR ligand binding domain that can compete with nativereceptors for ligand binding is a receptor blocking agent or ligandtrapping agent that falls within the scope of the present invention.

B. Protien Conjugates

With respect to the protein's half-life, one way to increase thecirculation half-life of a protein is to ensure a reduction in theclearance of the protein, in particular via renal clearance andreceptor-mediated clearance. This may be achieved by conjugating theprotein to a chemical moiety which is capable of increasing the apparentsize, thereby reducing renal clearance and increasing the in vivohalf-life. Furthermore, attachment of a chemical moiety to the proteinmay effectively block proteolytic enzymes from physical contact with theprotein, thus preventing degradation by non-specific proteolysis.Polyethylene glycol (PEG) is one such chemical moiety that has been usedin the preparation of therapeutic protein products. Recently, G-CSFmolecule modified with a single, N-terminally linked 20 kDa PEG group(Neulastam) was approved for sale in the United States. This PEGylatedG-CSF molecule has been shown to have an increased half-life compared tonon-PEGylated G-CSF and thus may be administered less frequently thancurrent G-CSF products, but it does not reduce the duration ofneutropenia significantly compared to non-PEGylated G-CSF.

Polyethylene glycol (PEG) modification is important for pharmaceuticaland biotechnological applications. PEGylation (the covalent attachmentof PEG) leads for example to shielding of antigenic or immunogenicepitopes. Moreover, it reduces receptor-mediated uptake by thereticuloendothelial system or prevents recognition and degradation byproteolytic enzymes. PEGylation of proteins has been shown to increasetheir bioavailability by reducing the renal filtration.

The term “conjugate” is intended to indicate a heterogeneous moleculeformed by the covalent attachment of one or more polypeptides, typicallya single polypeptide, to one or more non-polypeptide moieties such aspolymer molecules, lipophilic compounds, carbohydrate moieties ororganic derivatizing agents. The term covalent attachment means that thepolypeptide and the non-polypeptide moiety are either directlycovalently joined to one another, or else are indirectly covalentlyjoined to one another through an intervening moiety or moieties, such asa bridge, spacer, or linkage moiety or moieties. Preferably, theconjugate is soluble at relevant concentrations and conditions, i.e.,soluble in physiological fluids such as blood. Compositions and methodsfor preparing a conjugate of the invention are described in U.S. Pat.No. 6,831,158, which is incorporated herein by reference in itsentirety. The methods described in U.S. Pat. No. 6,831,158 are directedto conjugation of G-CSF, but can be readily adapted to conjugation ofthe sGPCRs of the present invention.

The “polymer molecule” is a molecule formed by covalent linkage of twoor more monomers. The term “polymer” may be used interchangeably withthe term “polymer molecule”. The term is intended to cover carbohydratemolecules including carbohydrate molecules attached to the polypeptideby in vivo N- or O-glycosylation, such molecule is also referred to as“an oligosaccharide moiety”. Except where the number of polymermolecule(s) is expressly indicated every reference to “a polymer”, “apolymer molecule”, “the polymer” or “the polymer molecule” contained ina polypeptide of the invention or otherwise used in the presentinvention shall be a reference to one or more polymer molecule(s).

The term “attachment group” is intended to indicate an amino acidresidue group of the polypeptide capable of coupling to the relevantnon-polypeptide moiety. For instance, for polymer conjugation, inparticular to PEG, a frequently used attachment group is the ε-aminogroup of lysine or the N-terminal amino group. Other polymer attachmentgroups include a free carboxylic acid group (e.g., that of theC-terminal amino acid residue or of an aspartic acid or glutamic acidresidue), suitably activated carbonyl groups, oxidized carbohydratemoieties and mercapto groups. Useful attachment groups and theirmatching non-peptide moieties are exemplified in Table 2. TABLE 2Attachment Example of non-peptide Conjungation method/ Group Amino Acidmoiety activated PEG Reference —NH₂ N-terminal Lys, Polymer, e.g., PEGwith mPEG-SPA Shearwater Corp. Arg, His amide or imine group TresylatedmPEG Delgado et al., 1992. —COOH C-terminal Asp Polymer, e.g., PEG withmPEG-Hz Shearwater Corp. and Glu ester or amide group in vitro couplingOligosaccharide moiety —SH Cys Polymer, e.g. PEG, with PEGvinylsullphone Shearwater Corp. disulfide, maleimide or PEG-maleimideDelgado et al., 1992 vinyl sulfone group In vitro couplingOligosaccharide moiety —OH Ser, Thr, —OH, lys Oligosaccharide moiety Invivo O-linked PAG with ester, ether, glycosylation carbamate, carbonate—CONH₂ Asn as part of an Oligosaccharide moiety In vivo N-glycosylationN-glycosylation Polymer, e.g. PEG site Aromatic Phe, Tyr, Trp,Oligosaxxharide moiety In vitro coupling Yan and Wold, 1984 —CONH₂ GlnAldehyde Oxidized Polymer, e.g. PEG PEGylation Andresz et al., 1978Ketone oligosaccharide PEG hydroxide WO 92/16655 WO 00/23114 GuanidinoArg Oligosaccharide moiety In vitro coupling Lunblad and Noyes, Chemicalreagents for protein modification, CRC Press Imidazole HisOligosaccharide moiety In vitro coupling Lunblad and Noyes, ringChemical reagents for protein modification, CRC Press

C. Site-Specific Mutagenesis

In one embodiment, amino acid sequence variants of a polypeptide can beprepared. These may, for instance, be minor sequence variants ofpolypeptides that arise due to natural variation within the populationor they may be homologs found in other species. They also may besequences that do not occur naturally but that are sufficiently similarthat they function similarly and/or elicit an immune response thatcross-reacts with natural forms of the polypeptide. Sequence variantscan be prepared by standard methods of site-directed mutagenesis such asthose described below.

Amino acid sequence variants of the polypeptide can be substitutional,insertional, or deletion variants. Deletion variants lack one or moreresidues of the native protein which are not essential for function orimmunogenic activity, and are exemplified by the variants of a receptorlacking a transmembrane sequence.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide such as stabilityagainst proteolytic cleavage or immunogenicity. Substitutions preferablyare conservative, that is, one amino acid is replaced with one ofsimilar shape and charge. Conservative substitutions are well known inthe art and include, for example, the changes of: alanine to serine;arginine to lysine; asparagine to glutamine or histidine; aspartate toglutamate; cysteine to serine; glutamine to asparagine; glutamate toaspartate; glycine to proline; histidine to asparagine or glutamine;isoleucine to leucine or valine; leucine to valine or isoleucine; lysineto arginine; methionine to leucine or isoleucine; phenylalanine totyrosine, leucine or methionine; serine to threonine; threonine toserine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine;and valine to isoleucine or leucine.

Insertional variants include fusion proteins such as those used to allowrapid purification of the polypeptide and also can include hybridproteins containing sequences from other proteins and polypeptides. Forexample, an insertional variant could include portions of the amino acidsequence of a polypeptide from one species, together with portions ofthe homologous polypeptide from another species. Other insertionalvariants can include those in which additional amino acids areintroduced within the coding sequence of the polypeptide, for example aprotease cleavage site(s) may be introduced.

Modification and changes may be made in the structure of apolynucleotide and still obtain a functional molecule that encodes aprotein or polypeptide with desirable characteristics. The following isa discussion based upon changing the amino acids of a protein to createan equivalent, or even an improved, second-generation molecule. Theamino acid changes may be achieved by changing the codons of the DNAsequence, according to the following data.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological activity certain amino acid substitutions can bemade in a protein sequence still obtain a protein with like properties.It is thus contemplated by the inventors that various changes may bemade in the DNA sequences of genes, mRNA or polynucleotides withoutappreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982). TABLE 3 Amino AcidsCodons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Asparticacid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUCUUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine IleI AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUUMethionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCGCCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGUSerine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACUValine Val V GUA GUG GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

It is accepted that the relative hydropathic character of the amino acidcontributes to the secondary structure of the resultant protein, whichin turn defines the interaction of the protein with other molecules, forexample, enzymes, substrates, receptors, DNA, antibodies, antigens, andthe like. It is known in the art that certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA. Ingeneral, the technique of site-specific mutagenesis is well known in theart. The technique typically employs a bacteriophage vector that existsin both a single stranded and double stranded form. Typical vectorsuseful in site-directed mutagenesis include vectors such as the M13phage. These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site-directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

The preparation of sequence variants of a GPCR, including but not limtedto sCRFR2α, polynucleotide using site-directed mutagenesis is providedas a means of producing potentially useful species, i.e., species withaltered ligand binding properties that include an increased affinity fora particular ligand, and is not meant to be limiting, as there are otherways in which sequence variants of nucleic acids may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

D. Expression and Purification of Polypeptides

The polynucleotides of the invention, in particular 100, 150, 200 250,300, 400, 450, 500, 550 or more contiguous nucleotides of the DNAencoding a GPCR, a family B GPCR, a family B1 GPCR, or a polynucleotidethat is 70, 75, 80, 85, 90, 95, 98, or 100% identical to the sequencespecified in the accompaning sequence listing, e.g., SEQ ID NO:1, 3, 5,7, 9, 11, 13, or 14 can be expressed as encoded peptides or proteins. Ina particular aspect the DNA encodes all or part of a GPCR extracellulardomain and in particular an amino terminal extracellular domain. Theengineering of DNA segment(s) for expression in a prokaryotic oreukaryotic system may be performed by techniques generally known tothose of skill in recombinant expression. It is believed that virtuallyany expression system may be employed in the expression of the claimednucleic acid sequences.

In certain embodiments, the present invention concerns novelcompositions comprising at least one proteinaceous molecule, such assGPCR, asCRFR, or a sCRFR2. As used herein, a “proteinaceous molecule,”“proteinaceous composition,” “proteinaceous compound,” “proteinaceouschain” or “proteinaceous material” generally refers, but is not limitedto, a protein of greater than about 200 amino acids or the full lengthendogenous sequence translated from a gene; a polypeptide of greaterthan about 100 amino acids; and/or a peptide of from about 3 to about100 amino acids. All the “proteinaceous” terms described above may beused interchangeably herein. Furthermore, these terms may be applied tofusion proteins as well.

In certain embodiments the size of the at least one proteinaceousmolecule may comprise, but is not limited to, about or at least 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 or greater aminomolecule residues, and any range derivable therein, particularly 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or morecontiguous amino acid sequences of such lengths of a GPCR, a family BGPCR, a family B1 GPCR, or SEQ ID NO:2, 4, 6, 8, 10, 12 or 15, includingthe full length of SEQ ID NO:4, 8, 12, or 15. Both cDNA and genomicsequences are suitable for eukaryotic expression, as the host cell willgenerally process the genomic transcripts to yield functional mRNA fortranslation into protein.

As used herein, the terms “engineered” and “recombinant” cells areintended to refer to a cell into which an exogenous DNA segment orpolynucleotide, such as a cDNA or polynucleotide has been introduced.Therefore, engineered cells are distinguishable from naturally occurringcells which do not contain a recombinantly introduced exogenous DNAsegment or gene. Engineered cells are thus cells having a gene or genesintroduced through the hand of man. Recombinant cells include thosehaving an introduced cDNA or genomic DNA, and may also include genespositioned adjacent to a promoter not naturally associated with theparticular introduced gene.

To express a recombinant protein or polypeptide, whether mutant orwild-type, in accordance with the present invention one would prepare anexpression vector that comprises one of the claimed isolated nucleicacids under the control of one or more promoters. To bring a codingsequence “under the control of” a promoter, one positions the 5′ end ofthe translational initiation site of the reading frame generally betweenabout 1 and 50 nucleotides “downstream” of (i.e., 3′ of) the chosenpromoter. The “upstream” promoter stimulates transcription of theinserted DNA and promotes expression of the encoded recombinant protein.This is the meaning of “recombinant expression” in the context usedhere.

Many standard techniques are available to construct expression vectorscontaining the appropriate nucleic acids andtranscriptional/translational control sequences in order to achieveprotein or peptide expression in a variety of host-expression systems.Cell types available for expression include, but are not limited to,bacteria, such as E. coli, B. subtilis, E. coli strain RR1, E. coliLE392, E. coli B, E. coli χ 1776 (ATCC No. 31537) as well as E. coliW3110 (F-, lambda-, prototrophic, ATCC No. 273325); bacilli such asBacillus subtilis; and other enterobacteriaceae such as Salmonellatyphimurium, Serratia marcescens, and various Pseudomonas speciestransformed with recombinant phage DNA, plasmid DNA or cosmid DNAexpression vectors.

The polynucleotide or polynucleotide fragment encoding a polypeptide canbe inserted into an expression vector by standard subcloning techniques.In one embodiment, an E. coli expression vector is used that producesthe recombinant polypeptide as a fusion protein, allowing rapid affinitypurification of the protein. Examples of such fusion protein expressionsystems are the glutathione S-transferase system (Pharmacia, Piscataway,N.J.), the maltose binding-protein system (New England Biolabs,Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6xHissystem (Qiagen, Chatsworth, Calif.). Further useful vectors include pINvectors (Inouye et al., 1985); and pGEX vectors, for use in generatingglutathione S-transferase (GST) soluble fusion proteins. Other suitablefusion proteins are those with β-galactosidase, ubiquitin, or the like.

For expression in Saccharomyces, the plasmid YRp7, for example, iscommonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemperet al., 1980). This plasmid contains the trp1 gene, which provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977).The presence of the trp1 lesion as a characteristic of the yeast hostcell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolyticenzymes (Hess et al., 1968; Holland et al., 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination.

Other suitable promoters, which have the additional advantage oftranscription controlled by growth conditions, include the promoterregion for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization.

In addition to micro-organisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture including mammalian and insect cells (e.g., U.S. Pat. No.4,215,051).

Examples of useful mammalian host cell lines are VERO and HeLa cells,Chinese hamster ovary (CHO) cell lines, WI38, BHK, COS-7, 293, HepG2,NIH3T3, RIN and MDCK cell lines. In addition, a host cell may be chosenthat modulates the expression of the inserted sequences, or modifies andprocesses the gene product in the specific fashion desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the encodedprotein.

Specific initiation signals may also be required for efficienttranslation of the claimed isolated nucleic acid coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences.Exogenous translational control signals, including the ATG initiationcodon, may additionally need to be provided. One of ordinary skill inthe art would readily be capable of determining this need and providingthe necessary signals. It is well known that the initiation codon mustbe in-frame (or in-phase) with the reading frame of the desired codingsequence to ensure translation of the entire insert. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements or transcription terminators (Bittner et al., 1987).

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably expressconstructs encoding G-proteins may be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with vectors controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched medium, and then areswitched to a selective medium. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci,which in turn can be cloned and expanded into cell lines.

A number of selection systems may be used, including, but not limited,to the herpes simplex virus thymidine kinase (Wigler et al., 1977),hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., 1962)and adenine phosphoribosyltransferase genes (Lowy et al., 1980), in tk⁻,hgprt⁻ or aprt⁻ cells, respectively. Also, antimetabolite resistance canbe used as the basis of selection for dhfr, which confers resistance tomethotrexate (Wigler et al., 1980; O'Hare et al., 1981); gpt, whichconfers resistance to mycophenolic acid (Mulligan et al., 1981); neo,which confers resistance to the aminoglycoside G-418 (Colbere-Garapin etal., 1981); and hygro, which confers resistance to hygromycin.

Once the polynucleotide sequence coding a particular polypeptide hasbeen determined or engineered, the polynucleotide can be inserted intoan appropriate expression system. In this case, the inventorscontemplate a polynucleotide encoding a sGPCR ligand binding domainpolypeptide. The polynucleotide can be expressed in any number ofdifferent recombinant DNA expression systems to generate large amountsof the polypeptide product, which can then be purified and/or isolatedto be used as a therapeutic or to vaccinate animals to generateantisera, or in certain aspects of the invention as an antagonist ofGPCR ligand and/or GPCR activation. In further aspects, sGPCRs of theinvention can be used in methods to detect, screen, or identify ligands,receptors, or agonist and/or antagonist of GPCRs. A polynucleotide ofthe invention may be expressed to obtain a GPCR ligand binding domain, afamily B GPCR ligand binding domain, a family B1 GPCR ligand bindingdomain, a sCRFR ligand binding domain or a CRFR2 ligand binding domainpolypeptide comprising an amino acid sequence including all or part ofthe amino acid sequence as set forth in the sequence listing, e.g., SEQID NO:2, 4, 6, 8, 10, 12, or 15.

As an alternative to recombinant polypeptides, synthetic peptidescorresponding to the polypeptides of the invention can be prepared,including antigenic peptides. Such antigenic peptides are at least sixamino acid residues long, and may contain up to approximately 35residues. Automated peptide synthesis machines include those availablefrom Applied Biosystems (Foster City, Calif.). Use of such smallpeptides for vaccination typically requires conjugation of the peptideto an immunogenic carrier protein such as hepatitis B surface antigen,keyhole limpet hemocyanin or bovine serum albumin. Methods forperforming this conjugation are well known in the art.

1. Purification of Expressed Proteins

Further aspects of the present invention concern the purification forisolation, and in particular embodiments, the substantial purification,of a protein or peptide comprising all or part of a sGPCR ligand bindingdomain. The term “purified or isolated protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein, polypeptide or peptide is purified toany degree relative to its naturally-obtainable state, i.e., in thiscase, relative to its purity within a organism or tissue. A purified orisolated protein or peptide therefore also refers to a protein orpeptide, free from the environment in which it may naturally occur. Apurified or isolated protein or polypeptide may have a purity greaterthan or at least 70, 75, 80, 85, 90, 95, 98, or 99% purity.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedactivity. Where the term “substantially purified” is used, thisdesignation will refer to a composition in which the protein or peptideforms the major component of the composition, such as constituting about50% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity (e.g., binding affinity for GPCR ligand including, butnot limited to CRF or a ligand of the CRF family) of an active fraction,or assessing the number of polypeptides within a fraction by SDS/PAGEanalysis. A preferred method for assessing the purity of a fraction isto calculate the specific activity of the fraction, to compare it to thespecific activity of the initial extract, and to thus calculate thedegree of purity, herein assessed by a “-fold purification number.” Theactual units used to represent the amount of activity, which may includebinding activity or affinity, will, of course, be dependent upon theparticular assay technique chosen.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, polyethylene glycol, antibodiesand the like or by heat denaturation, followed by centrifugation;chromatography steps such as ion exchange, gel filtration, reversephase, hydroxylapatite, and/or affinity chromatography; isoelectricfocusing; gel electrophoresis; and combinations of such and othertechniques. As is generally known in the art, it is believed that theorder of conducting the various purification steps may be changed, orthat certain steps may be omitted, and still result in a suitable methodfor the preparation of a substantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater-fold purification than thesame technique utilizing a low pressure chromatography system. Methodsexhibiting a lower degree of relative purification may have advantagesin total recovery of protein product, or in maintaining the activity ofan expressed protein.

E. Preparation of Antibodies Specific for sGPCRs

For some embodiments, it will be desired to produce antibodies that bindwith high specificity to the protein product(s) of an isolated nucleicacid encoding for sGPCR, including but not limted to sCRFR2α. In certainaspects, an antibody preparation is contemplated that recognizes orbinds the c-terminus of a GPCR, particularly a splice variant such as asCRFR2α splice variant and thus can be used to distingush a sGPCRpolypeptide from a membrane associated receptor. Such antibodies may beused in any of a variety of applications known to those of skill in theart, including but not limited to: immunodetection methods,immunoprecipitation methods, ELISA assays, protein purification methods,etc. Means for preparing and characterizing antibodies are well known inthe art (See, e.g., Harlow and Lane, 1988, incorporated herein byreference).

Methods for generating polyclonal antibodies are well known in the art.Briefly, a polyclonal antibody is prepared by immunizing an animal withan antigenic composition and collecting antisera from that immunizedanimal. A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig, a horse, or a goat.Because of the relatively large blood volume of rabbits, a rabbit is apreferred choice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

Monoclonal antibodies (MAbs) may be readily prepared through use ofwell-known techniques, such as those exemplified in U.S. Pat. No.4,196,265, incorporated herein by reference. Typically, this techniqueinvolves immunizing a suitable animal with a selected immunogencomposition, e.g., a purified or partially purified expressed protein,polypeptide or peptide. The immunizing composition is administered in amanner that effectively stimulates antibody producing cells.

The animals are injected with antigen as described above. Followingimmunization, somatic cells with the potential for producing antibodies,specifically B lymphocytes (B cells), are selected for use in the MAbgenerating protocol. Often, a panel of animals will have been immunizedand the spleen of the animal with the highest antibody titer will beremoved and the spleen lymphocytes obtained by homogenizing the spleenwith a syringe.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and have enzymedeficiencies that render them incapable of growing in certain selectivemedia that support the growth of only the desired fused cells(hybridomas). Any one of a number of myeloma cells may be used, as areknown to those of skill in the art (Goding, 1986). For example, wherethe immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653,NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210; and U-266, GM 1500-GRG2, LICR-LON-HMy2 and UC729-6 are all usefulin connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Large amounts of the monoclonal antibodies of the present invention mayalso be obtained by multiplying hybridoma cells in vivo. Cell clones areinjected into mammals that are histocompatible with the parent cells,e.g., syngeneic mice, to cause growth of antibody-producing tumors.Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection.

In accordance with the present invention, fragments of a monoclonalantibody can be obtained by methods which include digestion with enzymessuch as pepsin or papain and/or cleavage of disulfide bonds by chemicalreduction. Alternatively, monoclonal antibody fragments encompassed bythe present invention can be synthesized using an automated peptidesynthesizer, or by expression of full-length polynucleotide or ofpolynucleotide fragments encoding all or part of Mab.

Antibody conjugates may be prepared by methods known in the art, e.g.,by reacting an antibody with an enzyme in the presence of a couplingagent such as glutaraldehyde or periodate. Conjugates with fluoresceinmarkers are prepared in the presence of these coupling agents or byreaction with an isothiocyanate. Conjugates with metal chelates aresimilarly produced. Other moieties to which antibodies may be conjugatedinclude radionuclides such as ³H, ¹²⁵I, ¹³¹I ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl,⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, and ^(99m)Tc. Radioactively labeledantibodies of the present invention can be produced according towell-known methods. For instance, antibodies can be iodinated by contactwith sodium or potassium iodide and a chemical oxidizing agent such assodium hypochlorite, or an enzymatic oxidizing agent, such aslactoperoxidase. Antibodies according to the invention may be labeledwith technetium-⁹⁹ by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column or bydirect labelling techniques, e.g., by incubating pertechnate, a reducingagent such as SnCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the antibody.

III. Nucleic Acids Encoding sGPCR Polypeptides

The present invention includes nucleic acids that encode all or part ofa sGPCR, such as but not limited to a GPCR, a family B GPCR, a family B1GPCR, a CRFR, or a CRFR2 polypeptide, and may include various nucleicacid sequences needed for delivery of the nucleic acid sequence as wellas the transcription and/or translation of the nucleic acid seqeunce.Nucleic acid molecules of the invention may include various contiguousstretches of the nucleic acid, for example about 10, 15, 17, 20, 25, 30,35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200,250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750,2000, 2100, including all or part of the full length nucleic acidsequences in the sequence listing, e.g., SEQ ID NO:1, 3, 5, 7, 9, 11,13, or 14, or polynucleotides of those GPCRs referenced herein,fragments thereof, mRNAs, or cDNAs comprising sequences described orreferenced herein, and mutants of each are contemplated. Alsocontemplated are molecules that are complementary to the above mentionedsequences and that bind to these sequences under high stringencyconditions. These probes will be useful in a variety of hybridizationembodiments, such as Southern and northern blotting.

Various probes and primers can be designed around the disclosednucleotide sequences. Primers may be of any length but, typically, are10-20 bases in length. In particular aspects, the probe or primer can beused to identify or screen for the presence of an alternatively splicedform of a GPCR, such as but not limited to the CRFR2 gene that includesan exon 5/exon 7 splice junction (may also be described as an exon3/exon 5 juction as it relates to CRFR2α transcrition). These probes orprimers may either hybridize unique sequence of the engineered nulceicacid or splice junction, or amplify a nucleic acid characteristic of theengineered nucleic acid or the splice junction. By assigning numericvalues to a sequence, for example, the first residue is 1, the secondresidue is 2, etc., an algorithm defining all primers can be proposed:n to n+ywhere n is an integer from 1 to the last number of the sequence and y isthe length of the primer minus one, where n+y does not exceed the lastnumber of the sequence. Thus, for a 10-mer, the probes correspond tobases 1 to 10, 2 to 11, 3 to 12 . . . and so on. For a 15-mer, theprobes correspond to bases 1 to 15, 2 to 16, 3 to 17 . . . and so on.For a 20-mer, the probes correspond to bases 1 to 20, 2 to 21, 3 to 22 .. . and so on.

In certain aspects the nucleic acid seqeunces of the invention may beused to encode various polypeptides described herein. In one embodimentof the present invention, the nucleic acid sequences may be used ashybridization probes or amplification primers. In certain embodiments,these probes and primers consist of oligonucleotide fragments. Suchfragments should be of sufficient length to provide specifichybridization to an RNA or DNA sample extracted from tissue. Thesequences typically will be 10-20 nucleotides, but may be longer. Longersequences, e.g., 40, 50, 100, 500 and even up to full length, arepreferred for certain embodiments.

The use of a hybridization probe of between 17 and 100 nucleotides inlength allows the formation of a duplex molecule that is both stable andselective. Molecules having complementary sequences over stretchesgreater than 20 bases in length are generally preferred, in order toincrease stability and selectivity of the hybrid, and thereby improvethe quality and degree of particular hybrid molecules obtained. One willgenerally prefer to design nucleic acid molecules having stretches of 20to 30 nucleotides, or even longer where desired. Such fragments may bereadily prepared by, for example, directly synthesizing the fragment bychemical means or by introducing selected sequences into recombinantvectors for recombinant production. Accordingly, the nucleotidesequences of the invention may be used for their ability to selectivelyform duplex molecules with complementary stretches of genes,polynucleotides or RNAs, or to provide primers for amplification of DNAor RNA from tissues. Depending on the application envisioned, one willdesire to employ varying conditions of hybridization to achieve varyingdegrees of selectivity of probe towards target sequence.

For applications requiring high selectivity, one will typically desireto employ relatively stringent or high stringency conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by about 0.02 M to about 0.10 MNaCl at temperatures of about 50° C. to about 70° C. Such highstringency conditions tolerate little, if any, mismatch between theprobe and the template or target strand, and would be particularlysuitable for isolating specific genes or detecting specific mRNAtranscripts. It is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans of detection, such as a fluorescent or radiolabel, for determininghybridization. A wide variety of appropriate indicator means are knownin the art, including fluorescent, radioactive, enzymatic or otherligands, such as avidin/biotin, which are capable of being detected.

For applications in which the nucleic acid segments of the presentinvention are incorporated into expression vectors, such as plasmids,cosmids or viral polynucleotides, these segments may be combined withother DNA sequences, such as promoters, polyadenylation signals,restriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis contemplated that a nucleic acid fragment of almost any length may beemployed, with the total length preferably being limited by the ease ofpreparation and use in the intended recombinant DNA protocol.

DNA segments encoding a specific polynucleotide may be introduced intorecombinant host cells and employed for expressing a sGPCR, such as butnot limited to a sCRFR2α polypeptide. Alternatively, through theapplication of genetic engineering techniques, subportions orderivatives of selected polynucleotides may be employed.

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid having asequence defining a product, such as but not limited to a productencoding a polypeptide, in which part or all of the nucleic acidsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. Thus, in certainembodiments, expression includes both transcription of a polynucleotideand translation of a RNA into a polypeptide product.

In preferred embodiments, the nucleic acid is under transcriptionalcontrol of a promoter. A “promoter” refers to a DNA sequence recognizedby the synthetic machinery of the cell, or introduced syntheticmachinery, required to initiate the specific transcription of apolynucleotide. The phrase “under transcriptional control” means thatthe promoter is in the correct location and orientation in relation tothe nucleic acid to control RNA polymerase initiation and expression ofthe polynucleotide. The term promoter will be used here to refer to agroup of transcriptional control modules that are clustered around theinitiation site for a RNA polymerase, in particular RNA polymerase II.In certain aspects, at least one module in each promoter functions toposition the start site for RNA synthesis. The best known example ofthis is the TATA box, but in some promoters lacking a TATA box, such asthe promoter for the mammalian terminal deoxynucleotidyl transferasegene and the promoter for the SV40 late genes, a discrete elementoverlying the start site itself helps to fix the place of initiation.

The particular promoter that is employed to control the expression of anucleic acid is not believed to be critical, so long as it is capable ofexpressing the nucleic acid in the targeted cell. Thus, where a humancell is targeted, it is preferable to position the nucleic acid codingregion adjacent to and under the control of a promoter that is capableof being expressed in a human cell. Generally speaking, such a promotermight include either a human or viral promoter.

In various other embodiments, the human cytomegalovirus (CMV) immediateearly gene promoter, the SV40 early promoter and the Rous sarcoma viruslong terminal repeat can be used to obtain high-level expression oftransgenes. The use of other viral or mammalian cellular or bacterialphage promoters which are well-known in the art to achieve expression ofa transgene is contemplated as well, provided that the levels ofexpression are sufficient for a given purpose. Severalelements/promoters, as described below, may be employed, in the contextof the present invention, to regulate the expression of apolynucleotide, such as a transgene. This list is not intended to beexhaustive of all the possible elements involved in the promotion oftransgene expression but, merely, to be exemplary thereof.

Any promoter/enhancer combination (as per the Eukaryotic Promoter DataBase EPDB) could also be used to drive expression of a polynucleotide.Use of a T3, T7 or SP6 cytoplasmic expression system is another possibleembodiment. Eukaryotic cells can support cytoplasmic transcription fromcertain bacterial promoters if the appropriate bacterial polymerase isprovided, either as part of the delivery complex or as an additionalgenetic expression construct. Use of the baculovirus system will involvehigh level expression from the powerful polyhedrin promoter.

Promoters include, but are not limited to Immunoglobulin Heavy Chain,Immunoglobulin Light Chain, T-Cell Receptor, HLA DQ α and DQ β,β-Interferon, Interleukin-2, Interleukin-2 Receptor, MHC Class II 5, MHCClass II HLA-DRα, β-Actin, Muscle Creatine Kinase, Prealbumin(Transthyretin), Elastase I, Metallothionein, Collagenase, Albumin Gene,α-Fetoprotein, α-Globin, β-Globin, c-fos, c-HA-ras, Insulin, Neural CellAdhesion Molecule (NCAM), α₁-Anti-trypsin, H2B (TH2B) Histone, Mouse orType I Collagen, Glucose-Regulated Proteins (GRP94 and GRP78), RatGrowth Hormone, Human Serum Amyloid A (SAA), Troponin I (TN I),Platelet-Derived Growth Factor, Duchenne Muscular Dystrophy, SV40,Polyoma, Retroviruses, Papilloma Virus, Hepatitis B Virus, HumanImmunodeficiency Virus, Cytomegalovirus, Gibbon Ape Leukemia Virus.

Various element (inducers) include, but are not limited to MT II(Phorbol Ester (TPA) Heavy metals); MMTV (Glucocorticoids, β-Interferon,poly(rI)X, poly(rc)); Adenovirus 5 E2 (E1a); c-jun (Phorbol Ester (TPA),H₂O₂); Collagenase (Phorbol Ester (TPA)); Stromelysin (Phorbol Ester(TPA), IL-1); SV40 (Phorbol Ester (TPA)); Murine MX Gene (Interferon,Newcastle Disease Virus); GRP78 Gene (A23187); α-2-Macroglobulin (IL-6);Vimentin (Serum); MHC Class I Gene H-2kB (Interferon); HSP70 (E1a, SV40Large T Antigen); Proliferin (Phorbol Ester-TPA); Tumor Necrosis Factor(FMA); and Thyroid Stimulating Hormone α Gene (Thyroid Hormone).

One will typically include a polyadenylation signal to effect properpolyadenylation of the transcript. The nature of the polyadenylationsignal is not believed to be crucial to the successful practice of theinvention, and any such sequence may be employed. Preferred embodimentsinclude the SV40 polyadenylation signal and the bovine growth hormonepolyadenylation signal, convenient and known to function well in varioustarget cells. Also contemplated is the inclusion of a terminator as anelement of an expression cassette. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

In various embodiments of the invention, an expression construct maycomprise a virus or engineered construct derived from a viral genome.The ability of certain viruses to enter cells via receptor-mediatedendocytosis and to integrate into the host cell genome and express viralgenes stably and efficiently have made them attractive candidates forthe transfer of foreign genes into mammalian cells (Ridgeway, 1988;Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).The first viruses used as vectors were DNA viruses including thepapovaviruses (simian virus 40, bovine papilloma virus, and polyoma)(Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway,1988; Baichwal and Sugden, 1986) and adeno-associated viruses.Retroviruses also are attractive gene transfer vehicles (Nicolas andRubenstein, 1988; Temin, 1986) as are vaccina virus (Ridgeway, 1988) andadeno-associated virus (Ridgeway, 1988). Such vectors may be used to (i)transform cell lines in vitro for the purpose of expressinG-proteins ofinterest or (ii) to transform cells in vitro or in vivo to providetherapeutic polypeptides in a gene therapy scenario.

In an alternative embodiment, the sGPCR encoding nucleic acids employedmay actually encode antisense constructs that hybridize, underintracellular conditions, to a sGPCR or other encoding nucleic acid. Theterm “antisense construct” is intended to refer to nucleic acids,preferably oligonucleotides, complementary to the base sequences of atarget DNA or RNA.

As used herein, the terms “complementary” means nucleic acid sequencesthat are substantially complementary over their entire length and havevery few base mismatches. For example, nucleic acid sequences of fifteenbases in length may be termed complementary when they have acomplementary nucleotide at thirteen or fourteen positions with only asingle mismatch. Naturally, nucleic acid sequences which are “completelycomplementary” will be nucleic acid sequences which are entirelycomplementary throughout their entire length and have no basemismatches.

A. Detection and Quantitation of Nucleic Acids

One embodiment of the instant invention comprises a method foridentification of sGPCR nucleic acid, such as but not limited to CRFR2αnucleic acids, in a biological sample by amplifying and detectingnucleic acids corresponding to sGPCR. The biological sample can be anytissue or fluid in which the polynucleotide might be present. Nucleicacid used as a template for amplification is isolated from cellscontained in the biological sample, according to standard methodologies(Sambrook et al., 1989). The nucleic acid may be fractionated or wholecell RNA.

Pairs of primers that selectively hybridize to nucleic acidscorresponding to sGPCR are contacted with the isolated nucleic acidunder conditions that permit selective hybridization. Once hybridized,the nucleic acid:primer complex is contacted with one or more enzymesthat facilitate template-dependent nucleic acid synthesis. Multiplerounds of amplification, also referred to as “cycles,” are conducteduntil a sufficient amount of amplification product is produced. Theamplification products may be detected. In certain applications, thedetection may be performed by visual means. Alternatively, the detectionmay involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of incorporated radiolabelor fluorescent label, or even via a system using electrical or thermalimpulse signals (Affymax technology; Bellus, 1994).

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and in Innis et al., 1990, each of which isincorporated herein by reference in its entirety. Polymerase chainreaction methodologies are well known in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPA No. 320 308, which is incorporated herein by referencein its entirety. U.S. Pat. No. 4,883,750 describes a method similar toLCR for binding probe pairs to a target sequence. Also, Qbeta Replicase,described in PCT Application No. PCT/US87/00880, may be used as stillanother amplification method in the present invention. An isothermalamplification method, in which restriction endonucleases and ligases areused to achieve the amplification of target molecules that containnucleotide 5′-[alpha-thio]-triphosphates in one strand of a restrictionsite may also be useful in the amplification of nucleic acids in thepresent invention, Walker et al., (1992), incorporated herein byreference in its entirety. Still further, Strand DisplacementAmplification (SDA) is another method of carrying out isothermalamplification of nucleic acids which involves multiple rounds of stranddisplacement and synthesis, i.e., nick translation. A similar method,called Repair Chain Reaction (RCR), involves annealing several probesthroughout a region targeted for amplification, followed by a repairreaction in which only two of the four bases are present. Targetspecific sequences can also be detected using a cyclic probe reaction(CPR). Still another amplification method described in GB ApplicationNo. 2 202 328, and in PCT Application No. PCT/US89/01025, each of whichis incorporated herein by reference in its entirety, may be used inaccordance with the present invention. Other nucleic acid amplificationprocedures include transcription-based amplification systems (TAS),including nucleic acid sequence based amplification (NASBA) and 3SR(Kwoh et al., 1989); PCT Application WO 88/10315, incorporated herein byreference in their entirety).

Following amplification, it may be desirable to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al., 1989.

Chromatographic techniques may be employed to effect separation. Thereare many kinds of chromatography which may be used in the presentinvention: adsorption, partition, ion-exchange and molecular sieve, andmany specialized techniques for using them including column, paper,thin-layer and gas chromatography (Freifelder, 1982).

IV. Methods for sGPCR Gene Expression

In one embodiment of the present invention, there are provided methodsfor increased sGPCR expression in a cell, such as but not limited tosCRFR2α expression. This is particularly useful where there is anaberration in the protein or protein expression is not sufficient fornormal function. This will allow for the alleviation of symptoms ofdisease experienced as a result of deficiency of sGPCR, hyperactivationof GPCR or an abundance of GPCR ligand.

The general approach to increasing sGPCR is to contact or administer toa cell, tissue, animal, or subject a sGPCR polypeptide. While it ispreferred that the protein may be delivered directly, a conceivableembodiment involves providing a nucleic acid encoding a sGPCRpolypeptide to the cell or neighboring cells. Following this provision,the sGPCR polypeptide is synthesized by the host cell's transcriptionaland translational machinery, as well as any that may be provided by theexpression construct. Cis-acting regulatory elements necessary tosupport the expression of the sGPCR polynucleotide will be provided, inthe form of an expression construct. It also is possible that expressionof virally-encoded sGPCR could be stimulated or enhanced, or theexpressed polypeptide be stabilized, thereby achieving the same orsimilar effect.

In order to effect expression of constructs encoding sGPCRpolynucleotides, the expression construct must be delivered by adelivery vector into a cell. One mechanism for delivery is via viralinfection, where the expression construct is encapsidated in a viralparticle which will deliver either a replicating or non-replicatingnucleic acid.

The ability of certain viruses to enter cells via receptor-mediatedendocytosis, to integrate into host cell genome and express viral genesstably and efficiently have made them attractive candidates for thetransfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolasand Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The firstviruses used as gene vectors were DNA viruses including thepapovaviruses (simian virus 40, bovine papilloma virus, and polyoma)(Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway,1988; Baichwal and Sugden, 1986). These have a relatively low capacityfor foreign DNA sequences and have a restricted host spectrum.Furthermore, their oncogenic potential and cytopathic effects inpermissive cells raise safety concerns. They can accommodate only up to8 kb of foreign genetic material but can be readily introduced in avariety of cell lines and laboratory animals (Nicolas and Rubenstein,1988; Temin, 1986).

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells; they can also be used as vectors. Other viral vectorsmay be employed as expression constructs in the present invention.Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988;Baichwal and Sugden, 1986; Coupar et al., 1988) adeno-associated virus(AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska,1984) and herpesviruses may be employed. They offer several attractivefeatures for various mammalian cells (Friedmann, 1989; Ridgeway, 1988;Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et. al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use, as discussed below.

In another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro, but it may be applied toin vivo use as well. Another embodiment of the invention fortransferring a naked DNA expression construct into cells may involveparticle bombardment. This method depends on the ability to accelerateDNA coated microprojectiles to a high velocity allowing them to piercecell membranes and enter cells without killing them (Klein et al.,1987). Several devices for accelerating small particles have beendeveloped. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force(Yang et al., 1990). The microprojectiles used have consisted ofbiologically inert substances such as tungsten or gold beads.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Other expression constructs which can be employed to deliver a nucleicacid encoding a sCRFR2α polynucleotide into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

V. Pharmaceuticals and Methods for the Treatment of Disease

In additional embodiments, the present invention concerns formulation ofone or more of the polynucleotide, polypeptide, and/or antibodycompositions disclosed herein in pharmaceutically-acceptable solutionsfor administration to a cell, tissue, animal, patient, or subject eitheralone, or in combination with one or more other modalities of therapy.

Aqueous pharmaceutical compositions of the present invention will havean effective amount of a sGPCR expression construct, an expressionconstruct that encodes a therapeutic gene along with sGPCR, or a sGPCRprotein and/or compound that modulates GPCR ligand activity orsensititvy, or other endocrine function. Such compositions generallywill be dissolved or dispersed in a pharmaceutically acceptable carrieror aqueous medium. An “effective amount,” for the purposes of therapy,is defined at that amount that causes a clinically measurable differencein the condition of the subject. This amount will vary depending on thesubstance, the condition of the patient, the type of treatment, etc.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce a significantadverse, allergic or other untoward reaction when administered to ananimal, or human. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredients, its use inthe therapeutic compositions is contemplated.

In addition to the compounds formulated for parenteral administration,such as those for intravenous or intramuscular injection, otherpharmaceutically acceptable forms include, e.g., tablets or other solidsfor oral administration; time release capsules; and any other formcurrently used, including creams, lotions, inhalants and the like.

The active compounds of the present invention will often be formulatedfor parenteral administration, e.g., formulated for injection via theintravenous, intramuscular, subcutaneous, or even intraperitonealroutes. The preparation of an aqueous composition that contains sGPCRalone or in combination with a conventional therapeutic agent as activeingredients will be known to those of skill in the art in light of thepresent disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for using to prepare solutions or suspensions upon the additionof a liquid prior to injection can also be prepared; and thepreparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In many cases, the form must be sterile and must be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms, such as bacteria and fungi.

The carrier also can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,with even drug release capsules and the like being employable.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 mL of isotonic NaCl solutionand either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” (1980)). Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject.

In certain aspects of the methods of the invention, the route thetherapeutic composition is administered may be by parenteraladministration. The parenteral administration may be intravenousinjection, subcutaneous injection, intramuscular injection,intramedullary injection, ingestion or a combination thereof. In certainaspects, the composition comprising sGPCR is administered from about 0.1to about 10 microgram/kg/body weight per dose. In certain aspects, thecomposition comprising sGPCR is administered from about 1 to about 5microgram/kg/body weight per dose. In certain aspects, the compositioncomprising sGPCR is administered from about 1.2 to about 3.6microgram/kg/body weight per dose. In certain aspects, the compositioncomprising sGPCR is administered from about 1.2 to about 2.4microgram/kg/body weight per dose. In preferred aspects, the amount ofsGPCR administered per dose may be about 0.1, about 0.2, about 0.3,about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6,about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9,about 3.0, abot 3.1, about 3.2, about 3.3, about 3.4, about 3 5, about3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2,about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, abotu4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5,about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8,about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1,about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4,about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0, ormore micrograms/kg/body.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

A. Alimentary Delivery

The term “alimentary delivery” refers to the administration, directly orotherwise, to a portion of the alimentary canal of an animal. The term“alimentary canal” refers to the tubular passage in an animal thatfunctions in the digestion and absorption of food and the elimination offood residue, which runs from the mouth to the anus, and any and all ofits portions or segments, e.g., the oral cavity, the esophagus, thestomach, the small and large intestines and the colon, as well ascompound portions thereof such as, e.g., the gastro-intestinal tract.Thus, the term “alimentary delivery” encompasses several routes ofadministration including, but not limited to, oral, rectal, endoscopicand sublingual/buccal administration. A common requirement for thesemodes of administration is absorption over some portion or all of thealimentary tract and a need for efficient mucosal penetration of thenucleic acid(s) so administered.

1. Oral Delivery

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal, patient,or subject. As such, these compositions may be formulated with an inertdiluent or with an assimilable edible carrier, or they may be enclosedin hard- or soft-shell gelatin capsule, or they may be compressed intotablets, or they may be incorporated directly with the food of the diet.

The active components may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al.,1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and5,792,451, each specifically incorporated herein by reference in itsentirety). The tablets, troches, pills, capsules and the like may alsocontain the following: a binder, as gum tragacanth, acacia, cornstarch,or gelatin; excipients, such as dicalcium phosphate; a disintegratingagent, such as corn starch, potato starch, alginic acid and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin may be added or a flavoring agent, such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar, or both. A syrup of elixir may contain the active componentsucrose as a sweetening agent methyl and propylparabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

2. Rectal Administration

Therapeutics administered by the oral route can often be alternativelyadministered by the lower enteral route, i.e., through the anal portalinto the rectum or lower intestine. Rectal suppositories, retentionenemas or rectal catheters can be used for this purpose and may bepreferred when patient compliance might a otherwise be difficult toachieve (e.g., in pediatric and geriatric applications, or when thepatient is vomiting or unconscious). Rectal administration may result inmore prompt and higher blood levels than the oral route, but theconverse may be true as well (Harvey, 1990). Because about 50% of thetherapeutic that is absorbed from the rectum will bypass the liver,administration by this route significantly reduces the potential forfirst-pass metabolism (Benet et al., 1996).

B. Parenteral Delivery

The term “parenteral delivery” refers to the administration of atherapeutic of the invention to an animal, patient or subject in amanner other than through the digestive canal. Means of preparing andadministering parenteral pharmaceutical compositions are known in theart (see, e.g., Avis, 1990).

C. Intraluminal Administration

Intraluminal administration, for the direct delivery of a therapeutic toan isolated portion of a tubular organ or tissue (e.g., such as anartery, vein, ureter or urethra), may be desired for the treatment ofpatients with diseases or conditions afflicting the lumen of such organsor tissues. To effect this mode of administration, a catheter or cannulais surgically introduced by appropriate means. After isolation of aportion of the tubular organ or tissue for which treatment is sought, acomposition comprising a therapeutic of the invention is infused throughthe cannula or catheter into the isolated segment. After incubation forfrom about 1 to about 120 minutes, during which the therapeutic is takenup or in contact with the cells of the interior lumen of the vessel, theinfusion cannula or catheter is removed and flow within the tubularorgan or tissue is restored by removal of the ligatures which effectedthe isolation of a segment thereof (Morishita et al., 1993). Therapeuticcompositions of the invention may also be combined with a biocompatiblematrix, such as a hydrogel material, and applied directly to vasculartissue in vivo.

D. Intraventricular Administration

Intraventricular administration, for the direct delivery of atherapeutic to the brain of a patient, may be desired for the treatmentof patients with diseases or conditions afflicting the brain. One methodto affect this mode of administration, a silicon catheter is surgicallyintroduced into a ventricle of the brain of a human patient, and isconnected to a subcutaneous infusion pump (Medtronic Inc., Minneapolis,Minn.) that has been surgically implanted in the abdominal region (Zimmet al., 1984; Shaw, 1993). The pump is used to inject the therapeuticand allows precise dosage adjustments and variation in dosage scheduleswith the aid of an external programming device. The reservoir capacityof the pump is 18-20 mL and infusion rates may range from 0.1 mL/h to 1mL/h. Depending on the frequency of administration, ranging from dailyto monthly, and the dose of drug to be administered, ranging from 0.01μg to 100 g per kg of body weight, the pump reservoir may be refilled at3-10 week intervals. Refilling of the pump may be accomplished bypercutaneous puncture of the self-sealing septum of the pump.

E. Intrathecal Drug Administration

Intrathecal drug administration, for the introduction of a therapeuticinto the spinal column of a patient may be desired for the treatment ofpatients with diseases of the central nervous system. To effect thisroute of administration, a silicon catheter may be surgically implantedinto the L3-4 lumbar spinal interspace of a human patient, and isconnected to a subcutaneous infusion pump which has been surgicallyimplanted in the upper abdominal region (Luer and Hatton, 1993; Ettingeret al., 1978; Yaida et al., 1995). The pump is used to inject thetherapeutic and allows precise dosage adjustments and variations in doseschedules with the aid of an external programming device. The reservoircapacity of the pump is 18-20 mL, and infusion rates may vary from 0.1mL/h to 1 mL/h. Depending on the frequency of drug administration,ranging from daily to monthly, and dosage of drug to be administered,ranging from 0.01 μg to 100 g per kg of body weight, the pump reservoirmay be refilled at 3-10 week intervals. Refilling of the pump isaccomplished by a single percutaneous puncture to the self-sealingseptum of the pump.

To effect delivery to areas other than the brain or spinal column viathis method, the silicon catheter is configured to connect thesubcutaneous infusion pump to, e.g., the hepatic artery, for delivery tothe liver (Kemeny et al., 1993).

F. Vaginal Delivery

Vaginal delivery provides local treatment and avoids first passmetabolism, degradation by digestive enzymes, and potential systemicside-effects. Vaginal suppositories (Block, Chapter 87 In: Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, pages 1609-1614) or topical ointments can be used toeffect this mode of delivery.

G. Liposome-, Nanocapsule-, and Microparticle-Mediated Delivery

In certain embodiments, the inventors contemplate the use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, or ananoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically-acceptable formulations of the nucleic acids orconstructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art (see for example, Couvreuret al., 1977; Lasic, 1998; which describes the use of liposomes andnanocapsules in the targeted antibiotic therapy for intracellularbacterial infections and diseases). Recently, liposomes were developedwith improved serum stability and circulation half-times (Gabizon andPapahadjopoulos, 1988; Allen and Choun, 1987; U.S. Pat. No. 5,741,516,specifically incorporated herein by reference in its entirety). Further,various methods of liposome and liposome like preparations as potentialdrug carriers have been reviewed (Takakura, 1998; Chandran et al., 1997;Margalit, 1995; U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213;5,738,868 and 5,795,587, each specifically incorporated herein byreference in its entirety).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

The fate and disposition of intravenously injected liposomes depend ontheir physical properties, such as size, fluidity, and surface charge.They may persist in tissues for h or days, depending on theircomposition, and half lives in the blood range from min to several h.Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow, and lymphoid organs.

Alternatively, the invention provides for pharmaceutically-acceptablenanocapsule formulations of the compositions of the present invention.Nanocapsules can generally entrap compounds in a stable and reproducibleway (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998;Douglas et al., 1987). To avoid side effects due to intracellularpolymeric overloading, such ultrafine particles (sized around 0.1 μm)should be designed using polymers able to be degraded in vivo.Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet theserequirements are contemplated for use in the present invention. Suchparticles may be are easily made, as described (Couvreur et al., 1980;1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry etal. , 1995 and U.S. Pat. No. 5,145,684, specifically incorporated hereinby reference in its entirety).

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. The present examples, along with thecells and methods described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses which are encompassed within the spirit of the invention as definedby the scope of the claims will occur to those skilled in the art.

A. Materials and Methods

Isolation of the mouse soluble CRFR2α cDNA. The soluble CRFR2α splicevariant was isolated in parallel with that of the mouse CRFR2α ortholog.PCR primers were designed based on the homology between known mammalianCRFR2 genes. The following oligonucleotide primers, 5′CCCCGAAGCTGCCCGACTGG 3′ (SEQ ID NO:16) (sense) and 5′GGAAGGCTGTAAAGGATGGAGAAG 3′ (SEQ ID NO:17) (antisense) were used toscreen cDNA prepared from mouse whole brain poly(A)+ RNA which wasreverse transcribed using oligo dT or random primers. PCR was performedat 62° C. for 35 cycles with 90 sec extension at 72° C. The amplifiedfragments were subcloned into pCRIITOPO vector (Invitrogen, Carlsbad,Calif.), sequenced, and found to encode the full-length CRFR2α novelsplice variant lacking exon six, sCRFR2α (Chen et al., 2005).

Semi-quantitative RT-PCR and Southern analysis. The following mouseperipheral and CNS tissues were dissected and directly subjected tototal RNA isolation as previously described (Chen et al., 2005): totalbrain, olfactory bulb, hypothalamus, cortex, cerebellum, hippocampus,midbrain, pons/medulla oblongata, spinal cord and pituitary. The cDNAproducts were used as templates for semi-quantitative and RT PCRanalysis using specific primers for CRFR2α, sCRFR2α and the ribosomalprotein S 16. The locations of the oligonuleotide primers at exons threeand seven result in the amplification of two products of 418 and 309corresponding to CRFR2α and sCRFR2α, respectively. Oligonucleotideprimers sequence and PCR conditions can be found in the supporting text.

Extracellular receptor kinase 1/2 (ERK1/2) assay. CATH.a cells wereequilibrated with DMEM supplemented with 1% (w/v) bovine serum albumin(BSA) for 6 hr and then stimulated with 0.1% DMEM/BSA (vehicle) or 10 nMUcn I in the presence or absence of 0.4 or 4 nM sCRFR2α diluted in 0.1%DMEM/BSA. Cells were harvested immediately and analyzed forphosphorylated ERK1/2-p42, 44, as previously described (Chen et al.,2005).

Transient transfections and luciferase assay. The HEK293T cells weretransfected with a luciferase reporter containing a fragment of the EVX1gene containing a potent CRE site. The cells were harvested and theluciferase reporter activity was assayed as previously described (Chenet al., 2005). Twenty hours posttransfection, cells were treated for 4 hwith vehicle or with Ucn 1 (0.0001-100 nM) in the presence or absence of0.1 nM sCRFR2α.

Radio-Immuno Assays (RIA). Antisera was raised in rabbits immunized witha synthetic peptide fragment encoding the unique C-terminal tail (aa113-143) of mouse sCRFR2α conjugated to Keyhole Limpet Hemocyanin usinga protocol previously described for inhibin subunits (Vaughan et al.,1989). The analog Tyr¹¹³ sCRFR2α (113-143) was radiolabelled with Na¹²⁵Iusing chloramine-T and purified by HPLC (Vaughan et al., 1989) for useas tracer in the HA. The procedure for sCRFR2α RIA was similar to thatpreviously described in detail for inhibin subunits (Vaughan et al.,2005). Briefly, anti-sCRFR2α was used at 1/300,000 final dilution andsynthetic sCRFR2α (113-143) was used as standard. Murine tissues wereacid extracted and partially purified using octadecyl silica cartridgesas described (Vaughan et al., 1989). Lyophilized samples were tested atthree to seven dose levels. Free tracer was separated from bound by theaddition of sheep anti-rabbit γ-globulins and 10% (wt/vol) polyethyleneglycol. The EC50 and minimum detectable dose for sCRFR2α are ˜5 pg and100 pg per tube, respectively.

Immunohistochemistry. Adult male C57B6J mice (Jackson Laboratories) andSprague-Dawley albino rats (Harlan Sprague-Dawley) were anesthetizedwith chloral hydrate (350 mg/kg, ip) and perfused with Zamboni'sfixative (Bittencourt et al., 1999), followed by 0-4 hr. post-fixation.Regularly spaced (1-in-4) series of 30 μm thick frontal sectionsthroughout the brain were prepared for nickel-enhancedavidin-biotin-immunoperoxidase localization of sCRFR2α-ir usingVectastain Elite reagents (Vector Laboratories, Burlingame, Calif.).Primary sCRFR2α antisera were adsorbed against the carrier, affinitypurified and used at a dilution of 1:2000. Specificity of immunostainingwas evaluated using primary antisera preincubated overnight at 4° C.with 0-300 μM synthetic immunogen. Labeling was also evaluated in mutantmice deficient in either or both CRFRs (Smith et al., 1998; Bale et al.2000). Detailed description of the fluorescence immunocytochemicalanalysis of COSM6 cells transfected with sCRFR2α can be found in thesupporting text.

Mammalian expression of sCRFR2α: A cDNA corresponding to amino acids1-143, modified by PCR to include a FLAG epitope following amino acid143, was subcloned into pSec-Tag2 HygroA (Invitrogen, Carlsbad, Calif.)and used for transfection of COSM6 cells as described (Perrin et al.,2001). After 4 days, the media was collected and sCRFR2α was enriched bypurification using FLAG-agarose (Sigma, St. Louis, Mo.) immunoaffinitychromatography. The protein was detected by immunoblot analysis usingeither the anti-FLAG antibody or the antibody generated to the uniquesCRFR2α C-terminus.

Bacterial expression of sCRFR2α: A cDNA corresponding to amino acids20-143 was generated by PCR using mCRFR2α as the template. The cDNA wassubcloned into pET-32a(+) (Novagen, La Jolla, Calif.) and the proteinpurified by S-protein affinity chromatography as described (Perrin etal., 2001). The protein was detected by immunoblot analysis using theantibody generated to the unique sCRFR2α C-terminus.

Radioreceptor assays. The soluble protein, purified either from COS M6cell media or E. coli was incubated in triplicate wells with[¹²⁵I-DTyr^(O)]-astressin and increasing concentration of unlabeledpeptides as described (Perrin et al., 2003).

B. Results

A cDNA transcript of smaller (˜100 bp) size was observed during theisolation of the mouse CRFR2α (Van Pett et al., 2000). This smallerfragment was isolated and found to encode a variant of CRFR2α bearing adeletion of exon six. Translation of the variant transcript predicts anovel 143 amino acid protein, sCRFR2α, comprising the majority of thefirst extracellular domain of CRFR2α followed by a unique 38 amino acidC terminus (FIG. 1A). Screening of GenBank showed homology of theC-terminus to no other protein. The genomic arrangement of the sCRFR2αis shown in FIG. 1B.

If the sCRFR2α mRNA is merely a product of splicing errors, it should bemuch less abundant than the correctly spliced RNA. In order to examinethis question, semi- quantitative RT-PCR followed by Southernhybridization analysis was used to compare the relative abundance ofCRFR2α and sCRFR2α mRNA in several brain regions. Total RNA preparedfrom mouse tissues was reverse-transcribed to generate cDNAs that wereused as templates for semi-quantitative RT-PCR analysis, followed bySouthern hybridization, using specific primers and probes for CRFR2α andsCRFR2α (FIG. 2). The oligonucleotide primer pair (located in exonsthree and seven) allowed the simultaneous amplification of both thesoluble form and the full-length membrane bound receptor in a singlereaction (FIG. 2A). The sCRFR2α is highly expressed in the olfactory,cortex, midbrain and the pituitary (FIGS. 2B and 2C). Lower levels ofexpression were found in the hippocampus, hypothalamus, pons, medullaand spinal cord (FIGS. 2B and 2C). As shown in FIG. 2, the abundance ofsCRFR2α mRNA is lower, but comparable, to that of CRFR2α mRNA. Thesequences of cDNA fragments from RT-PCR were found to encode a splicevariant of the mouse CRFR2α gene (FIG. 1A).

Computer analysis of the sequence predicted that the first 19 aminoacids serve as a putative signal peptide. Because the sequence containsno obvious sites for membrane attachment, the protein is hypothesized tobe secreted as a soluble form. To explore this hypothesis, the proteinwas expressed in COS M6 cells. Following purification from the media, aprotein band of ˜30 kD was visualized by immunoblot analyses usingeither anti-FLAG antiserum or the anti-sCRFR2α, an antiserum raisedagainst a synthetic peptide fragment encoding the unique C-terminal tailof sCRFR2α protein (aa 113-143) (FIG. 3A). The larger size of theprotein compared to that predicted from the cDNA is probably a result ofglycosylation.

In order to obtain a larger quantity of sCRFR2α, a protein lacking theputative signal peptide was expressed as a fusion protein in E. coli(Perrin et al., 2001). Following cleavage and purification, the proteinwas visualized (using the anti-sCRFR2α) by immunoblot analysis as anarrow band of size ˜20 kD. The anti-sCRFR2α serum detects the sCRFR2αproteins both in radioimmunoassay (FIG. 3B) as well as inimmonocytochemistry (FIG. 3C).

Immunohistochemical studies using anti-sCRFR2α serum revealed thedistribution of sCRFR2α-ir in rodent brain. The cellular distribution ofimmunolabeling for sCRFR2α-ir was widespread and conformed more closelyto the location of CRFR1 mRNA expression pattern than to that of CRFR2(FIGS. 4A-4F). The results described are from studies in mice; a similarpattern of labeling was observed in rats. Major sites of cellularexpression include mitral and tufted cells of the olfactory bulb, themedial septavdiagonal band complex, piriform cortex, substantia nigra,red nucleus, basolateral amygdaloid, deep cerebellar and dorsal columnnuclei, all of which are prominent sites of CRFR1 expression. Similar toCRFR1, sCRFR2α-ir cell bodies are numerous throughout isocortex,although the laminar distributions are only partly overlapping. Thus,while both CRFR1- and sCRFR2α-expressing cell bodies are numerous inlayer 2/3, the dominant cortical seat of CRFR1 expression is in layer 4,while that of sCRFR2α is in layer 5. Major sites of CRFR2 expression,including the lateral septal, midbrain raphe, ventromedial hypothalamicand medial amygdaloid nuclei were all lacking in sCRFR2α-stained cellbodies, although interestingly the latter two sites were among the fewinvested with labeled varicosities that the inventors take to berepresentative of sCRFR2α-ir terminal fields. The paraventricularnucleus of the hypothalamus also contained a presumed sCRFR2α-irterminal field of moderate density.

Labeling throughout the brain was blocked by pre-incubation of theantiserum with low micromolar concentrations (≧30 μM) of the sCRFR2α(113-143) peptide used as immunogen; competition with the correspondingpeptide predicted from the CRFR1 sequence did not interfere withimmunolabeling at concentrations as high as 3 mM. Further support forthe specificity of labeling are observations that allimmunolocalizations persisted in CRFR1- and/or CRFR2-deleted mice; notethat the targeting construct used for generating each of the existingreceptor-knockout lines would be expected to spare the sCRFR2α codingregion (Smith et al., 1998; Timpl et al., 1998; Bale et al., 2000).

In order to determine the presence of sCRFR2α-like ir in brain, a highlyspecific radioimmunoassay was developed using anti-sCRFR2α-and[¹²⁵I-Tyr¹¹³] sCRFR2α (113-143)] as the tracer. Tissue from mouse brainwas acid-extracted, partially purified on C18 cartridges and assayed atmultiple doses in the radioimmunoassay. The tissue extracts displaced[¹²⁵I-Tyr¹¹³] sCRFR2α (113-143)] bound to anti-sCRFR2α in adose-dependent manner (FIG. 4G). Highest levels of expression were foundin the olfactory bulb, hypothalamus, cortex and midbrain, all of whichcorrelate with the presence of ir cells and fibers, determined by theimmunohistochemical studies (FIG. 4). A putative soluble form of CRFR1(generated by deletion of exon 5) would comprise a different uniqueC-terminal sequence. A protein corresponding to that sequence did notdisplace [¹²⁵I-Tyr¹¹³] sCRFR2α(113-143) in the radioimmunoassay. Theseresults further confirm the existence of sCRFR2α protein in rodent CNS.

The interactions of the sCRFR2α with CRF family ligands were assessed byradioreceptor assay using competitive displacement of [¹²⁵I-DTyr⁰]-astressin bound to sCRFR2α. The soluble proteins, secreted by COSM6 cells or produced in bacteria, bind the agonists, Ucn 1 and CRF, aswell as the antagonist, astressin, with nanomolar affinities, whereas,the affinities for Ucn 2 and Ucn 3 are much lower (Table 2). TABLE 2Inhibitory binding constants, Ki (nM) for CRF ligands binding tosCRFR2αproteins. Protein CRF rUcn1 mUcn2 mUcn3 Astressin mam sCRFR2α  23(14-39) 6.6 (3.5-12)   113 (68-190) >200 6.7 (3.6-12)  bact sCRFR2α 14.8(9.2-24) 5.8 (2.5-13.3) 116 (85-158) >200 10 (7.9-12.5)Binding of CRF family members to sCRFR2αproteins purified from eitherCOS M6 cell media (mam sCRFR2α) or E. coli (bactsCRFR2α). See Methodsfor details.

To delineate the possible hnctions of sCRFR2α, the inventors studied itseffects on signaling by CRF family ligands. Both the mammalian andbacterially expressed sCRFR2α proteins inhibit, in a dose dependentmanner, the cAMP response to Ucn 1 and CRF in HEK293T cells transfectedwith mouse CRFR2α as measured by the CRE luciferase activity of the EVX1gene (FIG. 5A). Because the urocortins activate MAPK signaling (Brar etal., 2002), the inventors measured the ability of sCRFR2α to inhibit theactivation by Ucn 1 of ERK1/2-p42,44 in CATH.a cells, which endogenouslyexpress CRFR1 and CRFR2α. The sCRFR2α inhibits the induction ofphosphorylated ERK by Ucn 1 in CATH.a cells (FIG. 5B).

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

REFERENCES

The references listed below are incorporated herein by reference to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

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1. An isolated, soluble corticotropin releasing factor receptor type 2(sCRFR2).
 2. The sCRFR2 of claim 1, wherein the sCRFR2 comprises anamino terminal extracellular domain of a corticotropin releasing factorreceptor type
 2. 3. The sCRFR2 of claim 1, wherein the amino acidsequence of the sCRFR2 comprises an amino acid sequence encoded by exons3, 4, and 5 of the CRFR2 gene.
 4. The sCRFR2 of claim 1, wherein thesCRFR2 comprises an amino acid sequence of at least 50 amino acids thatare at least 70% similar to the amino acid sequence of SEQ ID NO:4, SEQID NO:8, or SEQ ID NO:
 12. 5. The sCRFR2 of claim 4, wherein the sCRFR2comprises an amino acid sequence of at least 50 amino acids that are atleast 90% similar to the amino acid sequence of SEQ ID NO:4, SEQ IDNO:8, or SEQ ID NO:12.
 6. The sCRFR2 of claim 5, wherein the sCRFR2comprises an amino acid sequence of at least 50 amino acids that are atleast 95% similar to the amino acid sequence of SEQ ID NO:4, SEQ IDNO:8, or SEQ ID NO:12.
 7. The sCRFR2 of claim 6, wherein the sCRFR2comprises an amino acid sequence an amino acid sequence of at least 50amino acids of SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:12.
 8. The sCRFR2of claim 1, wherein the isolated sCRFR2 further comprises an affinitytag, a label, a radionuclide, an enzyme, a fluorescent marker, achemiluminescent marker, an immunoglobulin domain or a combinationthereof.
 9. The sCRFR2 of claim 8, wherein the isolated sCRFR2 furthercomprises an affinity tag.
 10. The sCRFR2 of claim 8, wherein theisolated sCRFR2 further comprises a fluorescent marker.
 11. The sCRFR2of claim 8, wherein the isolated sCRFR2 further comprises animmunoglobulin domain.
 12. The sCRFR2 of claim 11, wherein the sCRFR2comprises an immunoglobulin Fc domain.
 13. The s sCRFR2 of claim 1,further comprising a leader sequence.
 14. The sCRFR2 of claim 1, whereinthe sCRFR2 is conjugated to a polymer.
 15. The sCRFR2 of claim 14wherein the polymer is polyethylene glycol (PEG).
 16. An isolatednucleic acid encoding at least 50 consecutive amino acids of a sCRFR2.17. The nucleic acid of claim 16, further comprising a promoter operablycoupled to the nucleic acid encoding the sCRFR2.
 18. The nucleic acid ofclaim 17, wherein the nucleic acid is an expression cassette.
 19. Thenucleic acid of claim 18, wherein the expression cassette is comprisedin an expression vector.
 20. The nucleic acid of claim 19, wherein theexpression vector is a linear nucleic acid, a plasmid expression vector,or a viral expression vector.
 21. The nucleic acid of claim 19, whereinthe expression vector is operably coupled to a delivery vector.
 22. Thenucleic acid of claim 21, wherein the delivery vector is a liposome, apolypeptide, a polycation, a lipid, a bacterium, or a virus.
 23. Amethod of modulating activity of a G-protein coupled receptor (GPCR)comprising administering an effective dose of a sCRFR2 to a subject inneed thereof, wherein binding of a GPCR ligand to a cell surface GPCR isreduced.
 24. The method of claim 23, where the ligand is corticotropinreleasing factor (CRF), urocortin 1, urocortin 2, urocortin 3, orstresscopin.
 25. The method of claim 23, wherein administering is byingestion, injection, endoscopy, or perfusion.
 26. The method of claim25, wherein administering is by injection.
 27. The method of claim 26,wherein injection is intravenous injection, intramuscular injection,subcutaneous injection, intradermal injection, intracranial injection orintraperitoneal injection.
 28. The method of claim 23, wherein thesubject is human.
 29. The method of claim 23, further comprisingtreating a disorder resulting from hyperactivation of a GPCR orhypersecretion of GPCR ligand.
 30. The method of claim 29, wherein thedisorder is type II diabetes.
 31. The method of claim 29, wherein thedisorder is an type II diabetes, insulin sensitivity, anxiety-relateddisorders; a mood disorders; bipolar disorders; post-traumatic stressdisorder; inflammatory disorders; chemical dependencies and addictions;gastrointestinal disorders; or skin disorders.
 32. The method of claim31, wherein the anxiety-related disorder is generalized anxiety or themood disorder is depression.
 33. The method of claim 31, wherein thegastrointestinal disorder is irratable bowel syndrome.